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Modulating Collagen I Expression in Fibroblasts by CRISPR-Cas9 Base Editing of the Collagen 1A1 Promoter.
Fibrotic diseases, contributing to a significant portion of global mortality, highlight the need for innovative therapies. This study explores a novel approach to disrupt the expression of collagen by using adenine base editing to target Col1a1, a key gene driving both fibrosis and cancer metastasis. Editing Col1a1 in fibroblasts demonstrated 18% editing efficiency. An analysis of a specific clone harboring a CCAAT-to-CCGGA mutation in the Col1a1 promoter revealed reduced collagen production. Notably, when wild-type fibroblasts were cultured on the Col1a1-edited matrix, no compensatory collagen upregulation was detected, suggesting a lack of feedback mechanism in fibroblasts. Furthermore, the matrix derived from edited fibroblasts did not support the growth of MCF-7 cancer cells. These findings suggest that Col1a1 gene editing holds promise as a potential therapeutic strategy for fibrotic diseases. Further investigation is warranted to fully elucidate the implications of these findings for fibrosis and cancer.
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CRISPR/Cas systems: opportunities and challenges for crop breeding.
Increasing crop production to meet the demands of a growing population depends largely on crop improvement through new plant-breeding techniques (NPBT) such as genome editing. CRISPR/Cas systems are NPBTs that enable efficient target-specific gene editing in crops, which is supposed to accelerate crop breeding in a way that is different from genetically modified (GM) technology. Herein, we review the applications of CRISPR/Cas systems in crop breeding focusing on crop domestication, heterosis, haploid induction, and synthetic biology, and summarize the screening methods of CRISPR/Cas-induced mutations in crops. We highlight the importance of molecular characterization of CRISPR/Cas-edited crops, and pay special attentions to emerging highly specific genome-editing tools such as base editors and prime editors. We also discuss future improvements of CRISPR/Cas systems for crop improvement.
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Programmable Single and Multiplex Base-Editing in Bombyx mori Using RNA-Guided Cytidine Deaminases.
Genome editing using standard tools (ZFN, TALEN, and CRISPR/Cas9) rely on double strand breaks to edit the genome. A series of new CRISPR tools that convert cytidine to thymine (C to T) without the requirement for DNA double-strand breaks was developed recently and quickly applied in a variety of organisms. Here, we demonstrate that CRISPR/Cas9-dependent base editor (BE3) converts C to T with a high frequency in the invertebrate Bombyx mori silkworm. Using BE3 as a knock-out tool, we inactivated exogenous and endogenous genes through base-editing-induced nonsense mutations with an efficiency of up to 66.2%. Furthermore, genome-scale analysis showed that 96.5% of B. mori genes have one or more targetable sites that can be edited by BE3 for inactivation, with a median of 11 sites per gene. The editing window of BE3 reached up to 13 bases (from C1 to C13 in the range of gRNA) in B. mori Notably, up to 14 bases were substituted simultaneously in a single DNA molecule, with a low indel frequency of 0.6%, when 32 gRNAs were co-transfected. Collectively, our data show for the first time that RNA-guided cytidine deaminases are capable of programmable single and multiplex base editing in an invertebrate model.
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Establishment and rescue of fibroblast cell lines carrying a nonsense mutation of RB1 by CRISPR-based base editing.
Pathogenic variants of the RB1 gene have commonly been found in many cancer types, including retinoblastoma. Nonsense mutations are the most common mutation type in retinoblastoma; however, few cell lines mimic nonsense mutations in the RB1 gene that are commonly observed in patients. Here, we established retinoblastoma-like cell lines carrying mono- and bi-allelic nonsense mutations in the RB1 gene. We introduced the R552X mutation using target activation-induced cytidine deaminase base editing and successfully constructed cell lines carrying mono- and bi-allelic mutations. The model cell lines showed decreased RB1 expression at both the mRNA and protein levels, and increased cell proliferation. Furthermore, we rescued the nonsense mutation in the RB1 gene in model cell lines by converting stop codon 552 to tryptophan using an adenine base editor. This approach may be applicable for establishing cell lines with pathogenic variants found in patients and suggests a strong potential for the application of gene editing as a therapeutic strategy.
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RNA in Therapeutics: CRISPR in the Clinic.
The advent of the CRISPR-Cas genome editing platform has greatly enhanced the capabilities of researchers in many areas of biology. Its use has also been turned to the development of therapies for genetic diseases and to the enhancement of cell therapies. This review describes some recent advances in these areas.
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Generation of a multi-herbicide-tolerant alfalfa by using base editing.
We present the first report on base editing in alfalfa. Specifically, we showed edited alfalfa with tolerance to both sulfonylurea- and imidazolinone-type herbicides.
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sgBE: a structure-guided design of sgRNA architecture specifies base editing window and enables simultaneous conversion of cytosine and adenosine.
We present a base editing system, in which base editors are attached to different sites of sgRNA scaffold (sgBE). Each independent sgBE has its own specific editing pattern for a given target site. Among tested sgBEs, sgBE-SL4, in which deaminase is attached to the last stem-loop of sgRNA, yields the highest editing efficiency in the window several nucleotides next to the one edited by BE3. sgBE enables the simultaneous editing of adenine and cytosine. Finally, in order to facilitate in vivo base editing, we extend our sgBE system to an AAV-compatible Cas9, SaCas9 (Staphylococcus aureus), and observe robust base editing.
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Base editing treats SMA in mice.
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Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination.
Acetogenic bacteria are rising in popularity as chassis microbes for biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome and the potential for off-target events. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux toward improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general.
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Internally inlaid SaCas9 base editors enable window specific base editing.
Rationale: Base editors composed of catalytic defective Cas9 and cytosine or adenosine deaminase are powerful tools to convert bases in a genome. However, the fixed and narrow editing window of current base editors has impeded their utility. To increase the scope and diversify the editing patterns is quite necessary. Methods and Results: We designed a subset of base editors derived from SaCas9 in which deaminase was inlaid into various locations of the SaCas9 protein. The resulting base editors were characterized with multiple genomic sites and were found to have distinct editing features to the N-terminal SaCas9 CBE (Sa-CBE-N). Among them, Sa-CBE-693, in which a cytosine deaminase was inserted between amino acids 693 and 694, showed an increased editing efficiency and a significantly expanded editing window ranging from bases 2-18. This feature enhanced the editing efficiency of BCL11A enhancer that contains multiple consensus bases in a 15-bp fragment. Another variant, Sa-CBE-125, displayed backward-shifted editing window, which we showed was particularly powerful in editing cytosines that were accompanied with unintended bystander cytosines at their 5' side. Additionally, these editors showed reduced Cas9 independent DNA off-target editing compared with Sa-CBE-N. Conclusion: Our inlaid base editors improved the targeting scope and diversified the editing pattern.
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