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A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.
Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.
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Classification of CRISPR/Cas system and its application in tomato breeding.
Remarkable diversity in the domain of genome loci architecture, structure of effector complex, array of protein composition, mechanisms of adaptation along with difference in pre-crRNA processing and interference have led to a vast scope of detailed classification in bacterial and archaeal CRISPR/Cas systems, their intrinsic weapon of adaptive immunity. Two classes: Class 1 and Class 2, several types and subtypes have been identified so far. While the evolution of the effector complexes of Class 2 is assigned solely to mobile genetic elements, the origin of Class 1 effector molecules is still in a haze. Majority of the types target DNA except type VI, which have been found to target RNA exclusively. Cas9, the single effector protein, has been the primary focus of CRISPR-mediated genome editing revolution and is an integral part of Class 2 (type II) system. The present review focuses on the different CRISPR types in depth and the application of CRISPR/Cas9 for epigenome modification, targeted base editing and improving traits such as abiotic and biotic stress tolerance, yield and nutritional aspects of tomato breeding.
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The Expanding Dissemination and Distribution Patterns of Diverse CRISPR Plasmids by Addgene.
CRISPR-based technologies have rapidly enabled the democratization of genome editing in academic institutions through distribution by Addgene over the past decade. Recently, several distribution milestones have been reached, with a collection of >15,000 plasmids deposited by >1,000 laboratories spanning ∼40 countries now shipped 300,000 times to ∼5,000 organizations traversing ∼100 countries. Yet, both deposits of and requests for CRISPR plasmids continue to rise for this disruptive technology. Distribution patterns revealed robust demand for three distinct classes of CRISPR effectors, namely nucleases (e.g., Cas9 and Cas12), modulators (deactivated CRISPR nucleases fused to transcriptional regulators and epigenome modifiers), and chimeric effectors (Cas proteins fused to enzymes carrying out other activities such as deamination, reverse transcription, transposition, and integration). Yearly deposits over the past decade are requested in near-even proportions, reflecting continuous technological development and requests for novel constructs. Though it is unclear whether the slowing rate of requests is inherent to a pandemic operational lag or a transition from emerging to mature technology, it is noteworthy that the relative proportion of requests from plasmids deposited in the previous year remains stable, suggesting robust development of novel tools concurrent with continued adoption of editing, base editing, prime editing, and more. Predictably, most requested plasmids are designed for mammalian genome manipulation, presumably for medical research and human health pursuits, reflecting investments in therapeutic applications. Concurrently, requests for plant and microbial constructs are on the rise, especially in regions of the world more reliant on local agricultural inputs and focused on food and feed applications, illustrating continued diversification of genome editing applications.
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Treatment of a Mouse Model of ALS by In Vivo Base Editing.
Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal disorder that can be caused by mutations in the superoxide dismutase 1 (SOD1) gene. Although ALS is currently incurable, CRISPR base editors hold the potential to treat the disease through their ability to create nonsense mutations that can permanently disable the expression of the mutant SOD1 gene. However, the restrictive carrying capacity of adeno-associated virus (AAV) vectors has limited their therapeutic application. In this study, we establish an intein-mediated trans-splicing system that enables in vivo delivery of cytidine base editors (CBEs) consisting of the widely used Cas9 protein from Streptococcus pyogenes. We show that intrathecal injection of dual AAV particles encoding a split-intein CBE engineered to trans-splice and introduce a nonsense-coding substitution into a mutant SOD1 gene prolonged survival and markedly slowed the progression of disease in the G93A-SOD1 mouse model of ALS. Adult animals treated by this split-intein CRISPR base editor had a reduced rate of muscle atrophy, decreased muscle denervation, improved neuromuscular function, and up to 40% fewer SOD1 immunoreactive inclusions at end-stage mice compared to control mice. This work expands the capabilities of single-base editors and demonstrates their potential for gene therapy.
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Design of Acetohydroxyacid Synthase Herbicide-Resistant Germplasm through MB-QSAR and CRISPR/Cas9-Mediated Base-Editing Approaches.
The development of herbicide-resistant germplasm is significant in solving the increasingly severe weed problem in crop fields. In this study, we, for the first time, rationally designed a predictable and effective approach to create herbicide-resistant germplasm by combining mutation-dependent biomacromolecular quantitative structure-activity relationship (MB-QSAR) and CRISPR/Cas9-mediated base-editing strategies. Our results showed that the homozygous P197F-G654D-G655S or P197F-G654N-G655S Arabidopsis plants exhibited high resistance to multiple acetohydroxyacid synthase-inhibiting herbicides, including chlorsulfuron, bispyribac-sodium, and flucarbazone-sodium. Additionally, the plants with the homozygous P197S mutant displayed increased susceptibility to bispyribac-sodium than the wild-type but more resistance to flumetsulam than other mutants. Besides, we found that the herbicide resistance levels of the gene-edited plants have a good correlation with MB-QSAR prediction.
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A chemically controlled Cas9 switch enables temporal modulation of diverse effectors.
CRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.
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[Advances in genome editing and lodging resistance in sugarcane].
Sugarcane (Saccharum spp.) is an important cash crop that provides about 90% of sugar and 40% of bioethanol in China. Due to its large genome and complicated genetic background, conventional breeding is difficult to achieve efficient genetic improvement of sugarcane. Genome editing is a disruptive technology in life sciences, enabling precise and efficient modification of target genes. From zinc-finger nucleases (ZFNs) to transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system and the derived base editing and prime editing, these technologies have greatly advanced genetic research and upgraded biological breeding. With the decoding of the sugarcane genome, genome editing has provided a new technical means for the genetic improvement of polyploid sugarcane. This article provides a comprehensive review of the trajectory of genome editing in plants, the optimization of the CRISPR/Cas system, the genetic transformation status of sugarcane, the development of sugarcane genomics, and the application of genome editing in sugarcane. It focuses on exploring the application prospects of genome editing in breeding lodging-resistant sugarcane varieties. This review aims to provide valuable references for promoting the use of genome editing in sugarcane breeding.
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Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1) to nick the target DNA strand.
The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9 (SpCas9) and its variants. The detailed mechanism remains unknown. Here, based on in vitro cleavage assays and base editing analysis, we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1). We also show that these nicks are made on the target DNA strand. These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets. This system provides a novel tool for achieving trait stacking in plants.
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Adenine base editor engineering reduces editing of bystander cytosines.
Adenine base editors (ABEs) catalyze specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target site. To reduce the cytosine editing activity, we engineered a commonly used adenosine deaminase, TadA7.10, and found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited tenfold reduced cytosine deamination activity. The D108Q mutation also reduces cytosine deamination activity in two recently developed high-activity versions of ABE, ABE8e and ABE8s, and is compatible with V106W, a mutation that reduces off-target RNA editing. ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity and a substantially reduced adenine editing rate, yielding a TC-specific base editing tool for TC-to-TT or TC-to-TG conversions that broadens the utility of base editors.
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Selict-seq profiles genome-wide off-target effects in adenosine base editing.
Adenosine base editors (ABEs) facilitate A·T to G·C base pair conversion with significant therapeutic potential for correcting pathogenic point mutations in human genetic diseases, such as sickle cell anemia and β-thalassemia. Unlike CRISPR-Cas9 systems that induce double-strand breaks, ABEs operate through precise deamination, avoiding chromosomal instability. However, the off-target editing effects of ABEs remain inadequately characterized. In this study, we present a biochemical method Selict-seq, designed to evaluate genome-wide off-target editing by ABEs. Selict-seq specifically captures deoxyinosine-containing single-stranded DNA and precisely identifies deoxyadenosine-to-deoxyinosine (dA-to-dI) mutation sites, elucidating the off-target effects induced by ABEs. Through investigations involving three single-guide RNAs, we identified numerous unexpected off-target edits both within and outside the protospacer regions. Notably, ABE8e(V106W) exhibited distinct off-target characteristics, including high editing rates (>10%) at previously unreported sites (e.g. RNF2 and EMX1) and out-of-protospacer mutations. These findings significantly advance our understanding of the off-target landscape associated with ABEs. In summary, our approach enables an unbiased analysis of the ABE editome and provides a widely applicable tool for specificity evaluation of various emerging genome editing technologies that produce intermediate products as deoxyinosine.
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