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CRISPR-mediated editing of cis-regulatory elements in early mouse embryos: a tool for studying pluripotency gene regulation.
In brief Cis-regulatory elements and transcription factor binding motifs play crucial roles in regulating the spatial and temporal patterns of gene expression during development. This study tested the utility of CRISPR/Cas9 as a tool to interrogate the function of transcription factor AP2 gamma motifs in pluripotency gene expression during mouse preimplantation embryo development. Abstract Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) is a highly efficient tool that enables the generation of gene knockouts, knock-ins, and single base substitutions in a variety of organisms. Recently, we used CRISPR to examine the activity of cis-regulatory elements (CREs) in mouse preimplantation embryos. However, there is limited information on the feasibility of using CRISPR in preimplantation embryos to interrogate the function of select transcription factor (TF) binding motifs located within critical CREs in pluripotency genes. In the current study we employed CRISPR to disrupt TF AP2 gamma (TFAP2C) binding motifs located within key CREs involved in the regulation of Pou5f1 and Sox2 expression in early embryos. Microinjection of ribonucleoprotein complexes containing Cas9 and single guide RNAs (sgRNAs) targeting TFAP2C motifs located within a distal enhancer and proximal promoter substantially impaired Pou5f1 and Sox2 expression, respectively. Quantification of the editing efficiencies at each targeted CRE revealed that the targeting sgRNA sequences and the number of sgRNAs injected influenced the overall editing rates. Lastly, we investigated whether TFAP2C-induced activation of Sox2 expression in 2-cell embryos required TFAP2C motifs located within the Sox2 proximal promoter. CRISPR-mediated editing of these motifs diminished the activation of Sox2 expression. In summary, these findings indicate that CRISPR/Cas9 is a feasible approach for editing TF motifs in preimplantation embryos and provide evidence that TFAP2C directly contributes to Pou5f1 and Sox2 expression in preimplantation embryos.
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Development of a simple and locus-restricted DNA methylation editing system using direct fusion of a nickase-type SpCas9 and DNA methylation-related enzymes in Arabidopsis thaliana.
DNA methylation is an important epigenetic modification that regulates gene expression and supports genome stability. DNA methylation editing technology differs from conventional genome editing technology, which introduces mutations into genes, in that it enables changing gene expression without altering the base sequence. In this study, we attempted simple and locus-restricted DNA methylation editing in Arabidopsis thaliana using fusion proteins directly linking a nickase-type SpCas9 protein with DNA methylation-related enzymes. First, fusion of the human Ten-eleven translocation methyl cytosine dioxygenase 1 (TET1) catalytic domain (TET1cd) to nSpCas9 led to removing 5-methylcytosine in the FLOWERING LOCUS WA (FWA) promoter region of the wild-type plant, resulting in increased expression of the FWA gene and consequently, a late-flowering phenotype. Conversely, fusion of a mutant form of the bacterial DNA methyltransferase MQ1 (MQ1v) to nSpCas9 induced de novo DNA methylation in the fwa101-D mutant, in which the FWA promoter region is hypomethylated, and suppressed FWA gene expression, resulting in an early-flowering phenotype compared with the fwa101-D mutant. Of particular importance, our nSpCas9 system achieves targeted DNA methylation editing within a genomic window of ~10-20 kb. The nSpCas9 system features a compact and simplified vector structure due to the DNA methylation-related enzyme directly fusing to nSpCas9. Furthermore, sgRNA can be easily replaced, making it highly flexible. We propose a new method for targeted epigenome editing technology in plants, paving the way for innovative strategies in both basic research on epigenetics and crop development through epigenome editing.
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Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.
Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.
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CasY7: An optimized Cas12i system for enhanced genome editing in monocot crops.
The CRISPR-Cas12 family nucleases, particularly the Cas12i subtypes, are considered promising alternatives to Cas9 for genome editing in plants. We previously developed a new Cas12i variant, CasY7, which has been successfully applied in clinical trials; its performance in plants remains to be investigated. Initial testing in stable transgenic maize and rice showed that the codon-optimized CasY7 (pCasY7e1) achieved average editing efficiencies of 58.7% and 62.3% across five target sites, respectively, outperforming the typical Cpf1 (pCpf1) control that targets the same sites. To further enhance activity, we fused T5 exonuclease to CasY7 (pCasY7e2), which shifted mutation profiles toward larger deletions, and subsequently integrated an MS2 aptamer into the crRNA scaffold (pCasY7e3). The optimized pCasY7e3 system increased editing efficiencies to 87.7% in maize and 82.9% in rice-approximately 2.7-fold higher than pCpf1. We further demonstrated multiplexed editing in maize, generating biallelic dwarf mutants, and validated functionality in hexaploid wheat with editing efficiencies up to 58.8%. Overall, our comprehensive validation across 942 transgenic plants confirmed robust editing in maize, rice, and wheat, establishing CasY7 as a high-efficiency addition to the CRISPR toolkit.
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Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo.
Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5' end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5' end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5' end of the crRNA result in enhanced serum stability. Also, extending the 5' end of the crRNA by 59 nucleotides increases the delivery efficiency of Cpf1 RNP in cells and in vivo cationic delivery vehicles including polymer nanoparticle. Thus, 5' extension and chemical modification of the Cpf1 crRNA is an effective method for enhancing the gene editing efficiency of Cpf1 and its delivery in vivo.
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Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella phaffii.
There is growing interest in the use of nonmodel microorganisms as hosts for biopharmaceutical manufacturing. These hosts require genomic engineering to meet clinically relevant product qualities and titers, but the adaptation of tools for editing genomes, such as CRISPR-Cas9, has been slow for poorly characterized hosts. Specifically, a lack of biochemical characterization of RNA polymerase III transcription has hindered reliable expression of guide RNAs in new hosts. Here, we present a sequencing-based strategy for the design of host-specific cassettes for modular, reliable, expression of guide RNAs. Using this strategy, we achieved up to 95% gene editing efficiency in the methylotrophic yeast Komagataella phaffii. We applied this approach for the rapid, multiplexed engineering of a complex phenotype, achieving humanized product glycosylation in two sequential steps of engineering. Reliable extension of simple gene editing tools to nonmodel manufacturing hosts will enable rapid engineering of manufacturing strains tuned for specific product profiles and potentially decrease the costs and timelines for process development.
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CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion.
BACKGROUND: Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited. RESULTS: Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. CONCLUSIONS: Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.
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Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy.
In the absence of cell surface cancer-specific antigens, immunotherapies such as chimeric antigen receptor (CAR) T cells, monoclonal antibodies, or bispecific T cell engagers typically target lineage antigens. Currently, such immunotherapies are individually designed and tested for each disease. This approach is inefficient and limited to a few lineage antigens for which the on-target/off-tumor toxicities are clinically tolerated. Here, we sought to develop a universal CAR T cell therapy for blood cancers directed against the pan-leukocyte marker CD45. To protect healthy hematopoietic cells, including CAR T cells, from CD45-directed on-target/off-tumor toxicity while preserving the essential functions of CD45, we mapped the epitope on CD45 that is targeted by the CAR and used CRISPR adenine base editing to install a function-preserving mutation sufficient to evade CAR T cell recognition. Epitope-edited CD45 CAR T cells were fratricide resistant and effective against patient-derived acute myeloid leukemia, B cell lymphoma, and acute T cell leukemia. Epitope-edited hematopoietic stem cells (HSCs) were protected from CAR T cells and, unlike CD45 knockout cells, could engraft, persist, and differentiate in vivo. Ex vivo epitope editing in HSCs and T cells enables the safe and effective use of CD45-directed CAR T cells and bispecific T cell engagers for the universal treatment of hematologic malignancies and might be exploited for other diseases requiring intensive hematopoietic ablation.
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Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing.
Cas9-mediated gene editing is a powerful tool for addressing research questions in arthropods. Current approaches rely upon delivering Cas9 ribonucleoprotein (RNP) complex by embryonic microinjection, which is challenging, is limited to a small number of species, and is inefficient even in optimized taxa. Here we develop a technology termed Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to deliver Cas9 RNP to the arthropod germline by injection into adult female mosquitoes. We identify a peptide (P2C) that mediates transduction of Cas9 RNP from the female hemolymph to the developing mosquito oocytes, resulting in heritable gene editing of the offspring with efficiency as high as 0.3 mutants per injected mosquito. We demonstrate that P2C functions in six mosquito species. Identification of taxa-specific ovary-specific ligand-receptor pairs may further extend the use of ReMOT Control for gene editing in novel species.
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Programmable Genome-Editing Technologies as Single-Course Therapeutics for Atherosclerotic Cardiovascular Disease.
PURPOSE OF REVIEW: To establish genome editing as a promising therapeutic approach for the treatment and prevention of atherosclerotic cardiovascular disease. RECENT FINDINGS: Systemic delivery of a CRISPR adenine base editor using lipid nanoparticles demonstrated a near 90% reduction in circulating PCSK9 and over 60% reduction in blood LDL-C in nonhuman primates with the effects remaining durable at least 8 months following a single course. Preclinical proof-of-concept studies have elucidated the superior therapeutic potential of genome-editing approaches for the treatment of hyperlipidemia, thus substantiating their progression to clinical studies.
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