CRISPR-Cas9 Genome Editing Technology Research Articles

CRISPR-Cas9-mediated gene editing in human MPS I fibroblasts

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder (LSD). It is caused by mutations in the IDUA gene, which lead to the accumulation of the glycosaminoglycans dermatan and heparan sulfate. The CRISPR-Cas9 system is a new and powerful tool that allows gene editing at precise points of the genome, resulting in gene correction through the introduction and genomic integration of a wildtype sequence. In this study, we used the CRISPR-Cas9 genome editing technology to correct in vitro the most common mutation causing MPS I. Human fibroblasts homozygous for p.Trp402* (legacy name W402X) were transfected and analyzed for up to one month after treatment. IDUA activity was significantly increased, lysosomal mass was decreased, and next generation sequencing confirmed that a percentage of cells carried the wildtype sequence. As a proof of concept, this study demonstrates that CRISPR-Cas9 genome editing may be used to correct causative mutations in MPS I. List of abbreviationsUnlabelled TableCRISPRClustered Regularly Interspaced Short Palindromic RepeatsCasCRISPR-associatedERTenzyme replacement therapyHDRhomology-directed repairHSCThematopoietic stem cell transplantationIDUAalpha-L-iduronidaseLSDlysosomal storage disorderMPS IMucopolysaccharidosis type I

Inducing CCR5Δ32/Δ32 Homozygotes in the Human Jurkat CD4+ Cell Line and Primary CD4+ Cells by CRISPR-Cas9 Genome-Editing Technology

C-C chemokine receptor type 5 (CCR5) is the main co-receptor for HIV entry into the target CD4+ cells, and homozygous CCR5Δ32/Δ32 cells are resistant to CCR5-tropic HIV infection. However, the CCR5Δ32/Δ32 homozygous donors in populations are rare. Here we developed a simple approach to induce CCR5Δ32/Δ32 homozygotes through CRISPR-Cas9 genome-editing technology. Designing a pair of single-guide RNA targeting the flank region of the CCR5Δ32 mutation locus, we applied the CRISPR-Cas9 and lentiviral packaging system to successfully convert wild-type CCR5 into CCR5Δ32/Δ32 homozygotes in the human Jurkat CD4+ cell line and primary CD4+ cells, exactly the same as the naturally occurring CCR5Δ32/Δ32 mutation. The successful rate is up to 20% in Jurkat cells but less in primary CD4+ cells. The modified CCR5Δ32/Δ32 CD4+ cells are resistant to CCR5-tropic HIV infection. Whole-genome sequencing revealed no apparent off-target sites. This approach has the promise to promote HIV/AIDS therapy from the only cured unique Berlin patient to a routine autologous cell-based therapy.

Particle bombardment – mediated gene transfer and GFP transient expression in Setaria viridis

ABSTRACTSetaria viridis is one of the most important model grasses in studying monocot plant biology. Transient gene expression study is a very important tool in plant biotechnology, functional genomics, and CRISPR-Cas9 genome editing technology via particle bombardment. In this study, a particle bombardment-mediated protocol was developed to introduce DNA into Setaria viridis in vitro leaf explants. In addition, physical and biological parameters, such as helium pressure, distance from stopping screen to the target tissues, DNA concentration, and number of bombardments, were tested and optimized. Optimum concentration of transient GFP expression was achieved using 1.5 ug plasmid DNA with 0.6 mm gold particles and 6 cm bombardment distance, using 1,100 psi. Doubling the bombardment instances provides the maximum number of foci of transient GFP expression. This simple protocol will be helpful for genomics studies in the S. viridis monocot model.

FOXO Transcription Factors Rewire Metabolism in U87MG Glioblastoma Cells

Evolutionarily‐conserved FOXO transcription factors reside in the nucleus of glioblastoma and basal breast cancer cells that harbor constitutive PI3K Pathway output. Nuclear localization of FOXO factors in this setting challenges canonical PI3K models, as this pathway typically induces FOXO localization to the cytoplasm. Simultaneous PI3K activation and nuclear FOXO have also been observed in a set of diffuse large B cell lymphomas and embryonic stem cells. To interrogate the function of nuclear‐localized FOXO transcription factors in glioblastoma and basal breast cancer cell lines, we have constructed genetic models using CRISPR Cas9 genome editing technology. Our evidence indicates that FOXO factors drive metabolic changes in these cancers. FOXO disruption mutants showed increased mitochondrial membrane potential by TMRE staining and subsequent flow cytometric analyses. FOXO factors promote PEPCK gene expression to likely impact carbon utilization. These findings suggest that the nuclear localization of FOXO transcription factors causes metabolic rewiring in glioblastoma and basal breast cancer.Support or Funding InformationThis work was supported by HHMI 52007568 (N.V.), USDA Step 2 2015‐38422‐24061(A.L., R.G,), USDA H.S.I. 2016‐38422‐25760 (M.K. and E.M), NIH 1SC3GM11666901 (R.G.), UTRGV College of Sciences (COS) Seed Grant (M.K.), and NSF Advance 1209210 (M.K).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of the Activating Transcription Factor 5a (atf5a) gene in Hair Cell Development and Regeneration

Hair cells (HC) are sensory mechanoreceptors that are localized in sensory patches of the vertebrate inner ear. HC loss is the single leading cause of hearing and balance deficiencies. Mammals are unable to regenerate damaged HCs unlike lower vertebrates like birds, amphibians and fish. The two latter, also regenerate HCs in a superficial mechano‐sensory structure called the lateral line (LL) that allows them to sense water movements. Significant progress has been made in recent years by exploiting the experimental accessibility of the LL in a genetic tractable animal model: the zebrafish. The LL is composed of stereotypically distributed small sensory patches called neuromasts (NM). Each NM is a bulb‐shaped structure made of ~60 cells. It is externally delimited by ~10 mantle cells (MCs) which are forming an outer apical constricted ring through which protrude cilia from 12–15 centrally located HCs that are engulfed by 30–40 support cells (SC). Synchronized HC destruction will trigger a well described 3‐day regeneration program. New HCs are sequentially added from dividing and differentiating SCs. In parallel, other SCs proliferate replenishing their own stock, thus fitting the stem cell definition. Our lab has previously identified several genes with SC enriched expression. One of them is the activating transcription factor 5a (atf5a). It is a cAMP‐response element‐binding (CREB) transcription factor which was previously shown to have an important role in olfactory sensory neuron (OSN) differentiation in rodents, but was never described in the LL. To assess the role of this gene during OSN and HC development and regeneration, we first confirmed by in situ hybridization using an antisense probe directed against atf5a that it was expressed in both the olfactory pits and the LL. Next, we generated loss‐of‐function atf5a (KOs) using CRISPR‐Cas9 genome editing technology. So far, we have identified a 7 bp deletion (atf5a7D) that should represent a null allele, as reading frame shift is predicted to introduce an early stop codon in amino acid 93. We will present our preliminary results of the phenotypic analysis of homozygote (atf5a7D−/−) animals which we are focusing on the development and the regeneration of HCs in the LL and OSN in the nasal pits. Understanding the role of atf5a in the development and regeneration of these sensory tissues will help advance therapeutic regenerative approaches.Support or Funding InformationFunding: R25GM061838 MBRS‐RISE and NSF CREST Grant #HRD‐1137725This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of chimeric antigen receptors targeting T-cell malignancies using two structurally different anti-CD5 antigen binding domains in NK and CRISPR-edited T cell lines

ABSTRACTRelapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting. To circumvent this problem two approaches were investigated. First, a natural killer (NK) cell line, which does not express CD5, was used for CAR expression. Second, CRISPR-Cas9 genome editing technology was used to knockout CD5 expression in CD5-positive Jurkat T cells and in primary T cells, allowing for the use of CD5-negative T cells for CAR expression. Two structurally distinct anti-CD5 sequences were also tested, i) a traditional immunoglobulin-based single chain variable fragment (scFv) and ii) a lamprey-derived variable lymphocyte receptor (VLR), which we previously showed can be used for CAR-based recognition. Our results show i) both CARs yield comparable T-cell activation and NK cell-based cytotoxicity when targeting CD5-positive cells, ii) CD5-edited CAR-modified Jurkat T cells have reduced self-activation compared to that of CD5-positive CAR-modified T cells, iii) CD5-edited CAR-modified Jurkat T cells have increased activation in the presence of CD5-positive target cells compared to that of CD5-positive CAR-modified T cells, and iv) although modest effects were seen, a mouse model using the CAR-expressing NK cell line showed the scFv-CAR was superior to the VLR-CAR in delaying disease progression.

Hematopoietic stem cell gene therapy: progress and challenges

Hematopoietic stem cells possess the capacity of self-renewal and differentiation into all lineages of blood cells and have been recognized as one of the most ideal cells for gene therapy. Over the last decade, significant progress has been made in lentiviral vector-based hematopoietic stem cell gene therapy, and long-term success has been achieved in some clinical trials. The recent advent of the CRISPR-Cas9 genome editing technology has galvanized the field of gene therapy, and emerging on the horizon is targeted gene therapy by precise genome editing. However, to fulfill the vision of the second-generation hematopoietic stem cell gene therapy, further innovation is imperative to considerably increase the efficiency of precision genome editing in long-term hematopoietic stem cells.

The Smart Programmable CRISPR Technology: A Next Generation Genome Editing Tool for Investigators.

The present era is fast experiencing rapid innovation in the genome-editing technology. CRISPR Cas9-mediated targeted genetic manipulation is an easy, cost-effective and scalable method. As a result, it can be used for a broad range of targeted genome engineering. The main objective of the present review is to highlight the structural signature, classification, its mechanism and application from basic science to medicine and future challenges for this genome editing tool kit. The present review provides a brief description of the recent development of CRISPR-Cas9 genome editing technology. We discuss the paradigms shift for this next generation genome editing technology, CRISPR. The CRISPR structural significance, classification and its different applications are also being discussed. We portray the future challenges for this extraordinary genome in vivo editing tool. We also highlight the role of CRISPR genome editing in curing many diseases. Scientists and researchers are constantly looking one genome editing tool that is competent, simple and low-cost assembly of nucleases. It can target any particular site without any off-target mutations in the genome. The CRISPR-Cas9 has all of the above characteristics. The genome engineering technology may be a strong and inspiring technology meant for the next generation of drug development.

Abstract 1371: Isoprenylcysteine carboxylmethyltransferase (ICMT) function is critical for epithelial malignant transformation by mutant-Ras

Abstract Purpose Limited progress has been made in the development of direct inhibitors of Ras. Therefore, targeting isoprenylcysteine carboxylmethyltransferase (ICMT), the final enzyme in the prenylation pathway for Ras and other proteins containing the so-called CaaX-motif, remains an important strategy to inhibit Ras function in cancers. In this study, we investigate the role ICMT plays in the initiation of human epithelial cancers involving constitutively-active mutant Ras using genetic manipulation strategies. Methods Small T antigen and a shRNA against p53 (shp53) were sequentially expressed in human mammary epithelial cells immortalized by hTERT (HME1-hTERT) to create pre-malignant HME1-shp53 cells. All 4 isoforms of wild-type or mutant Ras, namely H-Ras, N-Ras, K-Ras-4A and K-Ras-4B, were independently introduced into HME1-shp53 cells to determine their transforming ability. Using CRISPR-Cas9 genome editing technology, ICMT-null HME1-shp53 cells were generated and mutant Ras isoforms were subsequently introduced; tumor formation abilities of thus generated cells were determined using soft-agar anchorage-independent colony formation assay and subcutaneous xenografts in mice. ICMT knock-out was also performed in MiaPaCa-2 and MDA-MB-231 cells, both expressing mutant K-Ras, and subcutaneous xenograft tumor formation in mice was measured. Results Pre-malignant HME1-shp53 cells can be transformed by all mutant Ras isoforms but not by any wild-type Ras. ICMT loss-of-function dramatically reduced anchorage-independent colony formation caused by all mutant Ras isoforms in HME1-shp53 cells, and in vivo xenograft formation was completely abolished. ICMT loss-of-function also dramatically reduced in vivo xenograft formation for mutant K-Ras-expressing MiaPaCa-2 and MDA-MB-231 naturally-occurring cancer cells. Conclusion ICMT function is essential for the malignant transformation of HME1 cells by all isoforms of Ras. Furthermore, ICMT function is also crucial for the maintenance of the malignant phenotype in mutant K-Ras-expresing cells such as MiaPaCa-2 and MDA-MB-231 cells. Citation Format: Hiu Yeung Lau, Jingyi Tang, Patrick J. Casey, Mei Wang. Isoprenylcysteine carboxylmethyltransferase (ICMT) function is critical for epithelial malignant transformation by mutant-Ras [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1371. doi:10.1158/1538-7445.AM2017-1371

Recent Advances in CRISPR-Cas9 Genome Editing Technology for Biological and Biomedical Investigations.

The Type II CRISPR-Cas9 system is a simple, efficient, and versatile tool for targeted genome editing in a wide range of organisms and cell types. It continues to gain more scientific interest and has established itself as an extremely powerful technology within our synthetic biology toolkit. It works upon a targeted site and generates a double strand breaks that become repaired by either the NHEJ or the HDR pathway, modifying or permanently replacing the genomic target sequences of interest. These can include viral targets, single-mutation genetic diseases, and multiple-site corrections for wide scale disease states, offering the potential to manage and cure some of mankind's most persistent biomedical menaces. Here, we present the developing progress and future potential of CRISPR-Cas9 in biological and biomedical investigations, toward numerous therapeutic, biomedical, and biotechnological applications, as well as some of the challenges within. J. Cell. Biochem. 119: 81-94, 2018. © 2017 Wiley Periodicals, Inc.

Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9.

Phages are biological entities found in every ecosystem. Although much has been learned about them in past decades, significant knowledge gaps remain. Manipulating virulent phage genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent lactococcal phages. Lactococcus lactis is a bacterium extensively used as a starter culture in various milk fermentation processes, and its phage sensitivity poses a constant risk to the cheese industry. The lactococcal phage p2 is one of the best-studied models for these virulent phages. Despite its importance, almost half of its genes have no functional assignment. CRISPR-Cas9 genome editing technology, which is derived from a natural prokaryotic defense mechanism, offers new strategies for phage research. Here, the well-known Streptococcus pyogenes CRISPR-Cas9 was used in a heterologous host to modify the genome of a strictly lytic phage. Implementation of our adapted CRISPR-Cas9 tool in the prototype phage-sensitive host L. lactis MG1363 allowed us to modify the genome of phage p2. A simple, reproducible technique to generate precise mutations that allow the study of lytic phage genes and their encoded proteins in vivo is described.

Generation of Genetically Modified Mice Using the CRISPR-Cas9 Genome-Editing System.

Genetically modified mice are extremely valuable tools for studying gene function and human diseases. Although the generation of mice with specific genetic modifications through traditional methods using homologous recombination in embryonic stem cells has been invaluable in the last two decades, it is an extremely costly, time-consuming, and, in some cases, uncertain technology. The recently described CRISPR-Cas9 genome-editing technology significantly reduces the time and the cost that are required to generate genetically engineered mice, allowing scientists to test more precise and bold hypotheses in vivo. Using this revolutionary methodology we have generated more than 100 novel genetically engineered mouse strains. In the current protocol, we describe in detail the optimal conditions to generate mice carrying point mutations, chromosomal deletions, conditional alleles, fusion tags, or endogenous reporters.

Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma

Sarcomas include some of the most aggressive tumors and typically respond poorly to chemotherapy. In recent years, specific gene fusion/mutations and gene over-expression/activation have been shown to drive sarcoma pathogenesis and development. These emerging genomic alterations may provide targets for novel therapeutic strategies and have the potential to transform sarcoma patient care. The RNA-guided nuclease CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein-9 nuclease) is a convenient and versatile platform for site-specific genome editing and epigenome targeted modulation. Given that sarcoma is believed to develop as a result of genetic alterations in mesenchymal progenitor/stem cells, CRISPR-Cas9 genome editing technologies hold extensive application potentials in sarcoma models and therapies. We review the development and mechanisms of the CRISPR-Cas9 system in genome editing and introduce its application in sarcoma research and potential therapy in clinic. Additionally, we propose future directions and discuss the challenges faced with these applications, providing concise and enlightening information for readers interested in this area.

Erratum to: Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology

Erratum to: Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology

CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani.

ABSTRACTThe prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani.

Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.

CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-induced mutations to the 5’ exons of candidate genes1–5, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. We show that the magnitude of negative selection reports the functional importance of individual protein domains of interest. A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.

A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans.

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.