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Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing.
Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.
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Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor.
Prime editors, novel genome-editing tools consisting of a CRISPR-Cas9 nickase and an engineered reverse transcriptase, can induce targeted mutagenesis. Nevertheless, much effort is required to optimize and improve the efficiency of prime-editing. Herein, we introduce two strategies to improve the editing efficiency using proximal dead sgRNA and chromatin-modulating peptides. We used enhanced prime-editing to generate Igf2 mutant mice with editing frequencies of up to 47% and observed germline transmission, no off-target effects, and a dwarf phenotype. This improved prime-editing method can be efficiently applied to cell research and to generate mouse models.
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Programmed cell death-1-modified pig developed using electroporation-mediated gene editing for in vitro fertilized zygotes.
Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs.
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CRISPR base editing and prime editing: DSB and template-free editing systems for bacteria and plants.
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) has been extensively exploited as a genetic tool for genome editing. The RNA guided Cas nucleases generate DNA double-strand break (DSB), triggering cellular repair systems mainly Non-homologous end-joining (NHEJ, imprecise repair) or Homology-directed repair (HDR, precise repair). However, DSB typically leads to unexpected DNA changes and lethality in some organisms. The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools. Hence, in this review, we focus on the non-DSB and template-free genome editing, i.e., base editing (BE) and prime editing (PE) in bacteria and plants. We first highlight the development of base and prime editors and summarize their studies in bacteria and plants. We then discuss current and future applications of BE/PE in synthetic biology, crop improvement, evolutionary engineering, and metabolic engineering. Lastly, we critically consider the challenges and prospects of BE/PE in PAM specificity, editing efficiency, off-targeting, sequence specification, and editing window.
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PlmCas12e (CasX2) cleavage of CCR5: impact of guide RNA spacer length and PAM sequence on cleavage activity.
Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from Streptococcus pyogenes and Staphylococcus aureus, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5-7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in CCR5 (CCR5-[Formula: see text]32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR/Cas. We determined that CCR5 cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the CCR5-[Formula: see text]32 mutation in haematopoietic stem cells.
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RNA-seq Analysis of the SCN1A-KO Model based on CRISPR/Cas9 Genome Editing Technology.
Dravet syndrome (DS) is a disease that is primarily caused by the inactivation of the SCN1A-encoded voltage-gated sodium channel alpha subunit (Nav1.1). In this study, we constructed an SCN1A gene knockout model using CRISPR/Cas9 genome editing technology to deprive the Nav1.1 function in vitro. With mRNA-seq analysis we found abundant gene changes after SCN1A knockout, which associated with various signaling pathways, such as cancer pathways, the PI3K-AKT signaling pathway, the MAPK signaling pathway, and pathways involved in HTLV-I infection. We also noticed changes in the spliceosome, decreased glycolytic capacity, disturbances in calcium signaling pathways, and changes in the potassium, sodium, chloride, and calcium plasma channels after SCN1A knockout. In this study, we have been the first time to discover these changes and summarize them here and hope it would provide some clue for the study of Nav1.1 in the nervous system.
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Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes.
Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.
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A critical view on using "life not worth living" in the bioethics of assisted reproduction.
This paper critically engages with how life not worth living (LNWL) and cognate concepts are used in the field of beginning-of-life bioethics as the basis of arguments for morally requiring the application of preimplantation genetic diagnosis (PGD) and/or germline genome editing (GGE). It is argued that an objective conceptualization of LNWL is largely too unreliable in beginning-of-life cases for deriving decisive normative reasons that would constitute a moral duty on the part of intending parents. Subjective frameworks are found to be more suitable to determine LNWL, but they are not accessible in beginning-of-life cases because there is no subject yet. Conceptual and sociopolitical problems are additionally pointed out regarding the common usage of clear case exemplars. The paper concludes that a moral requirement for the usage of PGD and GGE cannot be derived from the conceptual base of LNWL, as strong reasons that can be reliably determined are required to limit reproductive freedom on moral grounds. Educated predictions on prospective well-being might still be useful regarding the determination of moral permissibility of PGD and/or GGE. It is suggested that due to the high significance of subjective experience in the normativity of beginning-of-life bioethics, the discipline is called to more actively realize the inclusion of people with disabilities. This regards for instance research design, citation practices, and language choices to increase the accessibility of societal debates on the reproductive ethics of genetic technologies.
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Advances in CRISPR-Cas systems for human bacterial disease.
Prokaryotic adaptive immune systems called CRISPR-Cas systems have transformed genome editing by allowing for precise genetic alterations through targeted DNA cleavage. This system comprises CRISPR-associated genes and repeat-spacer arrays, which generate RNA molecules that guide the cleavage of invading genetic material. CRISPR-Cas is classified into Class 1 (multi-subunit effectors) and Class 2 (single multi-domain effectors). Its applications span combating antimicrobial resistance (AMR), targeting antibiotic resistance genes (ARGs), resensitizing bacteria to antibiotics, and preventing horizontal gene transfer (HGT). CRISPR-Cas3, for example, effectively degrades plasmids carrying resistance genes, providing a precise method to disarm bacteria. In the context of ESKAPE pathogens, CRISPR technology can resensitize bacteria to antibiotics by targeting specific resistance genes. Furthermore, in tuberculosis (TB) research, CRISPR-based tools enhance diagnostic accuracy and facilitate precise genetic modifications for studying Mycobacterium tuberculosis. CRISPR-based diagnostics, leveraging Cas endonucleases' collateral cleavage activity, offer highly sensitive pathogen detection. These advancements underscore CRISPR's transformative potential in addressing AMR and enhancing infectious disease management.
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Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.
CRISPR-Cas base-editor technology enables targeted nucleotide alterations, and is being increasingly used for research and potential therapeutic applications1,2. The most widely used cytosine base editors (CBEs) induce deamination of DNA cytosines using the rat APOBEC1 enzyme, which is targeted by a linked Cas protein-guide RNA complex3,4. Previous studies of the specificity of CBEs have identified off-target DNA edits in mammalian cells5,6. Here we show that a CBE with rat APOBEC1 can cause extensive transcriptome-wide deamination of RNA cytosines in human cells, inducing tens of thousands of C-to-U edits with frequencies ranging from 0.07% to 100% in 38-58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, and 5' and 3' untranslated region mutations. We engineered two CBE variants bearing mutations in rat APOBEC1 that substantially decreased the number of RNA edits (by more than 390-fold and more than 3,800-fold) in human cells. These variants also showed more precise on-target DNA editing than the wild-type CBE and, for most guide RNAs tested, no substantial reduction in editing efficiency. Finally, we show that an adenine base editor7 can also induce transcriptome-wide RNA edits. These results have implications for the use of base editors in both research and clinical settings, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
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