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Precise A∙T to G∙C base editing in the allotetraploid rapeseed (Brassica napus L.) genome.
Rapeseed is an important source of oilseed crop in the world. Achieving genetic improvement has always been the major goal in rapeseed production. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Nowadays, Cas-base editing acts as an efficient tool to mediate single-base substitution at the target site. In this study, four adenine base editors (ABE) were modified to achieve adenosine base editing at different genome sites in allotetraploid Brassica napus. We designed 18 small guide RNAs to target phytoene desaturase (PDS), acetolactate synthase (ALS), CLAVATA3 (CLV3), CLV2, TRANSPARENT TESTA12 (TT12), carotenoid isomerase (CRTISO), designated de-etiolated-2 (DET2), BRANCHED1 (BRC1), zeaxanthin epoxidase (ZEP) genes, respectively. Among the four ABE systems, pBGE17 had the highest base-editing efficiency, with an average editing efficiency of 3.51%. Target sequencing results revealed that the editing window ranged from A5 to A8 of the protospacer-adjacent motif (PAM) sequence. Moreover, the ABEmax-nCas9NG system with NG PAM was developed, with a base-editing efficiency of 1.22%. These results revealed that ABE system developed in this study could efficiently induce A to G substitution and the ABE-nCas9NG system could broaden editing window in oilseed rape.
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Base editing the mammalian genome.
Base editing is a powerful technology that enables programmable conversion of single nucleotides in the mammalian genome. Base editors consist of a partially active Cas9 nuclease (Cas9D10A) tethered to a natural or synthetic DNA modifying enzyme. Though only recently described, BE has already shown enormous potential for basic and translational research, allowing the creation or repair of disease alleles in a variety of cell types and model organisms. In the past 2 years, a vast array of new and modified base editor variants have been described, expanding the flexibility and usefulness of the approach. Though simple in concept, effective implementation of base editing requires an understanding of the advantages and limitations of each of these tools. Here, we provide an overview of the concepts of DNA base editing, and discuss the recent progress toward the development of optimized base editing systems for mammalian cells. In addition, we highlight key technical aspects of designing and executing BE experiments, and provide detailed experimental examples of successful base editing in cell lines and organoids to help guide the effective use of these tools for genome modification.
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Cytosine base editor-DNA binding domain fusions for editing window modulation in the RNP format.
Base editing technologies allow for the precise and efficient installation of defined nucleotide substitutions into a target genome without the introduction of double strand breaks or DNA templates. Here we describe two recombinant, protein format cytosine base editors (CBEs) that efficiently catalyze the installation of cytosine-to-thymine edits, termed "Flexible" and "Precision." Flexible exhibits a wide editing window, while Precision uses a fused single-stranded DNA binding protein to narrow the editing window, lowering the risk of editing multiple cytosine residues at the target site. We show that co-transfection with uracil glycosylase inhibitor protein increases the proportion of substitutions that are C-to-T and the ratio of C-to-T editing to indel formation, thus reducing undesired editing outcomes. We use in vitro editing assays to characterize our editors and show a preference for cytosine residues preceded by thymine (TpC dinucleotides) and unmethylated cytosine residues.
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Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.
Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.
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Transgene-free genome editing in poplar.
Precise gene-editing methods are valuable tools to enhance genetic traits. Gene editing is commonly achieved via stable integration of a gene-editing cassette in the plant's genome. However, this technique is unfavorable for field applications, especially in vegetatively propagated plants, such as many commercial tree species, where the gene-editing cassette cannot be segregated away without breaking the genetic constitution of the elite variety. Here, we describe an efficient method for generating gene-edited Populus tremula × P. alba (poplar) trees without incorporating foreign DNA into its genome. Using Agrobacterium tumefaciens, we expressed a base-editing construct targeting CCoAOMT1 along with the ALS genes for positive selection on a chlorsulfuron-containing medium. About 50% of the regenerated shoots were derived from transient transformation and were free of T-DNA. Overall, 7% of the chlorsulfuron-resistant shoots were T-DNA free, edited in the CCoAOMT1 gene and nonchimeric. Long-read whole-genome sequencing confirmed the absence of any foreign DNA in the tested gene-edited lines. Additionally, we evaluated the CodA gene as a negative selection marker to eliminate lines that stably incorporated the T-DNA into their genome. Although the latter negative selection is not essential for selecting transgene-free, gene-edited Populus tremula × P. alba shoots, it may prove valuable for other genotypes or varieties.
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[Recent advances in CRISPR-related transposable elements].
A new wave of research has been inspired by the CRISPR-Cas system with respect to their application in genome editing. The CRISPR-Cas system can not only be applied in gene knockout and insertion, but also be used in base editing, transcriptional regulation and recombination of gene clusters. However, the low efficiency of homology-directed repair (HDR) limits its application. Unlike the CRISPR-Cas system, mobile genetic elements (MGE) can insert DNA fragments into cell chromosomes without the aid of HDR. Recently, it is reported that CRISPR-related transposable elements can guide targeted DNA insertion. Their transposition mechanisms and reprogramming abilities have brought novel opportunities to the development of this field. This review summarized the research progress and application development of natural CRISPR-related transposable elements in recent years, as well as the applications of fused dCas9-transposase. It proposed the application prospects and potential challenges of CRISPR-related transposable elements in the future, which provided a reference for the development direction of gene editing tools.
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Engineering CjCas9 for Efficient Base Editing and Prime Editing.
The CRISPR-Cas9 system has been applied for clinical applications of gene therapy. Most CRISPR-based gene therapies are derived from Streptococcus pyogenes Cas9, which is challenging to package into a single adeno-associated virus vector and limits its clinical applications. Campylobacter jejuni Cas9 (CjCas9) is one of the smallest Cas9 proteins. CjCas9-mediated base editing (CjBE) efficiency varies across genomic sites, while CjCas9-mediated prime editing (CjPE) efficiency is less than 5% on average. Here we developed enhanced cytosine base editors (enCjCBEs) and adenine base editors (enCjABEs) by engineered CjCas9P47K. We demonstrated the robust C-to-T conversion (70% on average) by enCjCBE or A-to-G conversion (76% on average) by enCjABE. Meanwhile, we applied the CjCas9P47K variant to generate enhanced CjPE (enCjPE), which increases the editing efficiency 17-fold at the PRNP site over wild-type CjPE. Fusing nonspecific DNA binding protein Sso7d to enCjCas9 and MS2 stem-loop RNA aptamer to the 3-terminal of cognate pegRNA resulted in 12% editing efficiency on average with a 24-fold increase over wild-type CjPE, and we termed it SsenCjPE. The SsenCjPE can also be combined with hMLH1dn to further increase the editing efficiency and MMLV RTaseΔRnH to reduce size. Finally, we introduced an additional mutation D829R into SsenCjPE and generated SsenCjPE-M2 with a 61-fold increase of PE efficiency over wild-type at the PRNP site. In summary, enCjBEs, SsenCjPEs, or SsenCjPE-M2 are compact Cas9-derived BE or prime editors in biological research or biomedical applications.
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CRISPR-Cas9-Based Genome Editing and Cytidine Base Editing in Acinetobacter baumannii.
Infections caused by drug-resistant Acinetobacter baumannii have posed a serious threat to global public health. However, genetic manipulation methods, the primary way to study pathogenesis and drug-resistance mechanisms, remain time consuming and inefficient. Here, we provide a detailed protocol for genetic manipulation, including gene deletion, insertion, and point mutation in A. baumannii using the platform. For complete details on the use and execution of this protocol, please refer to Wang et al. (2019).
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Applying gene editing to tailor precise genetic modifications in plants.
The ability to tailor alterations in genomes, including plant genomes, in a site-specific manner has been greatly advanced through approaches that reduced the complexity and time of genome sequencing along with development of gene editing technologies. These technologies provide a valuable foundation for studies of gene function, metabolic engineering, and trait modification for crop improvement. Development of genome editing methodologies began ∼20 years ago, first with meganucleases and followed by zinc finger nucleases, transcriptional activator-like effector nucleases and, most recently, clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas) (CRISPR/Cas), which is by far the most utilized method. The premise of CRISPR/Cas centers on the cleaving of one or both DNA strands by a Cas protein, an endonuclease, followed by mending of the DNA by repair mechanisms inherent in cells. Its user-friendly construct design, greater flexibility in targeting genomic regions, and cost-effective attributes have resulted in it being widely adopted and revolutionizing precise modification of the genomes of many organisms. Indeed, the CRISPR/Cas system has been utilized for gene editing in many plant species, including important food crops, such as maize, wheat, rice, and potatoes. This review summarizes the various approaches, including the most recent designs being used to make modifications from as small as a single-base-pair change to insertion of DNA fragments. On the gene expression level, strategies are presented that make it possible to knock out or modulate through activation and repression. Also discussed are prerequisites necessary for CRISPR/Cas-mediated editing as well as the current challenges.
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Base editing for reprogramming cyanobacterium Synechococcus elongatus.
Cyanobacteria can directly convert carbon dioxide (CO2) at the atmospheric level to biofuels, value-added chemicals and food products, making them ideal candidates to alleviate global climate change. Despite decades-long pioneering successes, the development of genome-editing tools, especially the CRISPR-Cas-based approaches, seems to lag behind other microbial chassis, slowing down the innovations of cyanobacteria. Here, we adapted and tailored base editing for cyanobacteria based on the CRISPR-Cas system and deamination. We achieved precise and efficient genome editing at a single-nucleotide resolution and demonstrated multiplex base editing in the model cyanobacterium Synechococcus elongatus. By using the base-editing tool, we successfully manipulated the glycogen metabolic pathway via the introduction of premature STOP codons in the relevant genes, building engineered strains with elevated potentials to produce chemicals and food from CO2. We present here the first report of base editing in the phylum of cyanobacteria, and a paradigm for applying CRISPR-Cas systems in bacteria. We believe that our work will accelerate the metabolic engineering and synthetic biology of cyanobacteria and drive more innovations to alleviate global climate change.
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