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Transient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells.
Precise gene editing in human pluripotent stem cells (hPSCs) holds great promise for studying and potentially treating human diseases. Both prime editing and base editing avoid introducing double strand breaks, but low editing efficiencies make those techniques still an arduous process in hPSCs. Here we report that co-delivering of p53DD, a dominant negative fragment of p53, can greatly enhance prime editing and cytosine base editing efficiencies in generating precise mutations in hPSCs. We further apply PE3 in combination with p53DD to efficiently create multiple isogenic hPSC lines, including lines carrying GBA or LRRK2 mutations associated with Parkinson disease and a LMNA mutation linked to Hutchinson-Gilford progeria syndrome. We also correct GBA and LMNA mutations in the patient-specific iPSCs. Our data show that p53DD improves PE3 efficiency without compromising the genome-wide safety, making it feasible for safe and routine generation of isogenic hPSC lines for disease modeling.
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CRISPR mediated gene editing for economically important traits in horticultural crops: progress and prospects.
Horticultural crops, with their cost-effectiveness, rich mineral and vitamin content, and high yield potential, have become indispensable worldwide for ensuring food and nutritional security. With the world's population on the rise and climate change becoming more prominent, it is crucial to focus on creating resilient, high-yielding crop varieties that can withstand the changing climate. Genetic improvement of different horticultural crops using conventional tools is both time-consuming and labourious. However, the breeding period can be cut short by adopting modern breeding techniques, including CRISPR/Cas-mediated genome editing. In the present review, we discuss the progress made so far through genome editing to improve several horticultural crops for various traits like stress resistance, morphology, nutritional attributes, quality, shelf life, male sterility, architecture and economic yield. We have also discussed the emerging CRISPR technologies like base editing, epigenome editing, CRE editing, transposon-based editing, prime editing etc., along with their pros and cons and the future prospects. The ethical considerations for commercialization and current regulatory frameworks for gene-edited products have also been discussed.
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High-efficiency and multiplex adenine base editing in plants using new TadA variants.
Recently reported adenine base editors (ABEs) exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding. However, editing efficiency varies significantly among different target sites. Here, we investigated the activities of three evolved E. coli adenosine deaminase TadA variants (TadA8e, TadA8.17, and TadA8.20) side-by-side in transgenic rice. We found that TadA8e outperforms TadA8.17 and TadA8.20, and induces efficient A-to-G conversion at all tested sites in the rice genome, including those that were uneditable by ABE7.10 in our previous experiments. Furthermore, V82S/Q154R mutations were incorporated into TadA8e, resulting in a new variant that we named TadA9. Our data show that TadA9 is broadly compatible with CRISPR/SpCas9, CRISPR/SpCas9-NG, and CRISPR/SpRY, as well as CRISPR/ScCas9 nickase systems, achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e. Finally, TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in weed control. Collectively, we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome. Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.
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Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells.
Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.
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Prime editing: current advances and therapeutic opportunities in human diseases.
Gene editing ushers in a new era of disease treatment since many genetic diseases are caused by base-pair mutations in genomic DNA. With the rapid development of genome editing technology, novel editing tools such as base editing and prime editing (PE) have attracted public attention, heralding a great leap forward in this field. PE, in particular, is characterized by no need for double-strand breaks (DSBs) or homology sequence templates with variable application scenarios, including point mutations as well as insertions or deletions. With higher editing efficiency and fewer byproducts than traditional editing tools, PE holds great promise as a therapeutic strategy for human diseases. Subsequently, a growing demand for the standard construction of PE system has spawned numerous easy-to-access internet resources and tools for personalized prime editing guide RNA (pegRNA) design and off-target site prediction. In this review, we mainly introduce the innovation and evolutionary strategy of PE systems and the auxiliary tools for PE design and analysis. Additionally, its application and future potential in the clinical field have been summarized and envisaged.
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Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3'-End-Truncated crRNAs.
Mismatch tolerance, a cause of the off-target effect, impedes accurate genome editing with the CRISPR/Cas system. Herein, we observed that oligonucleotide-directed single-base substitutions could be rarely introduced in the microbial genome using CRISPR/Cpf1-mediated negative selection. Because crRNAs have the ability to recognize and discriminate among specific target DNA sequences, we systematically compared the effects of modified crRNAs with 3'-end nucleotide truncations and a single mismatch on the genomic cleavage activity of FnCpf1 inEscherichia coli. Five nucleotides could be maximally truncated at the crRNA 3'-end for the efficient cleavage of the DNA targets of galK and xylB in the cells. However, target cleavage in the genome was inefficient when a single mismatch was simultaneously introduced in the maximally 3'-end-truncated crRNA. Based on these results, we assumed that the maximally truncated crRNA-Cpf1 complex can distinguish between single-base-edited and unedited targets in vivo. Compared to other crRNAs with shorter truncations, maximally 3'-end-truncated crRNAs showed highly efficient single-base substitutions (>80%) in the DNA targets of galK and xylB. Furthermore, the editing efficiency for the 24 bases in both galK and xylB showed success rates of 79 and 50%, respectively. We successfully introduced single-nucleotide indels in galK and xylB with editing efficiencies of 79 and 62%, respectively. Collectively, the maximally truncated crRNA-Cpf1 complex could perform efficient base and nucleotide editing regardless of the target base location or mutation type; this system is a simple and efficient tool for microbial genome editing, including indel correction, at the single-nucleotide resolution.
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Dynamics of single-base editing: Theoretical analysis.
Recent experimental advances led to the development of DNA base editors (BEs) with single-nucleotide precision, which is critical for future progress in various scientific and technological fields. The molecular mechanisms of single-base discrimination, however, remain poorly understood. Using a recently developed stochastic approach, we theoretically investigated the dynamics of single-base editing. More specifically, transient and mean times to edit "TC" motifs by cytosine BEs are explicitly evaluated for correct (target) and incorrect (bystander) locations on DNA. In addition, the effect of mutations on the dynamics of the single-base edition is also analyzed. It is found that for most ranges of parameters, it is possible to temporarily separate target and bystander products of base editing, supporting the idea of dynamic selectivity as a method of improving the precision of single-base editing. We conclude that to improve the efficiency of single-base editing, selecting the probability or selecting the time requires different strategies. Physical-chemical arguments to explain the observed dynamic properties are presented. The theoretical analysis clarifies some important aspects of the molecular mechanisms of selective base editing.
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STAGE: A compact and versatile TnpB-based genome editing toolkit for Streptomyces.
Streptomyces are naturally endowed with the capacity to produce a wide array of natural products with biomedical and biotechnological value. They have garnered great interest in synthetic biology applications given the abundance of uncharacterized biosynthetic gene clusters (BGCs). However, progress has been hindered by the limited availability of genetic tools for manipulating these bacteria. Several representative CRISPR-Cas systems have been established in Streptomyces to streamline experimental workflows and improve editing efficiency. Nevertheless, their broader applicability has been constrained by issues such as nuclease activity-related cytotoxicity and the large size of effector proteins. To address these challenges, we present Streptomyces-compatible TnpB-assisted genome editing (STAGE), a genetic toolkit based on ISDra2 TnpB, which is approximately one-third the size of Cas9 and enables precise, site-specific gene editing. We demonstrated that STAGE introduces genetic mutations with high efficiency and minimal off-target effects in two industrially important Streptomyces strains. Building on this platform, we developed STAGE-cBEST and STAGE-McBEST, enabling single and multiplexed C·G-to-T·A base editing, respectively, with editing efficiencies exceeding 75%. To further enhance performance, we engineered the ISDra2 TnpB system using an AI-assisted protein engineering framework, resulting in two variants that achieve nearly 100% genome editing efficiency. Additionally, through sequence homology analysis, we identified a TnpB ortholog from the same biological origin of ISDra2 TnpB, which also functions effectively as a gene editing tool. Our study establishes STAGE as a highly precise, programmable, and versatile genome editing platform for Streptomyces, paving the way for advanced genetic manipulation and synthetic biology applications in these industrially important bacteria.
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Development of a highly efficient base editing system for Lactobacilli to improve probiotics and dissect essential functions.
Lactobacilli play essential roles in the food industry and have a significant potential as probiotics and therapeutic agents. Genomic and genetic information has increasingly accumulated and been linked to their various functions, to which transgenic approaches are being performed to verify crucial genes. In order to reasonably develop more useful strains, beneficial traits need to be introduced into any given strains and enhanced or combined based on such genotype characterization. However, for practical use as probiotics or foods, organisms with transgene are hardly acceptable. Here, we have introduced the base editing Target-AID system specifically for Lactobacilli, enabling precise installation of point mutations without donor DNA and at multiple genomic loci simultaneously. Lactiplantibacillus plantarum has been successfully engineered to reduce production of imidazole propionate, which has been reported to be associated with type 2 diabetes by impairing glucose tolerance and insulin signaling. Additionally, this system enabled transient knock-out of an essential gene, such as one involved in cell division, resulting in severe filamentous cell phenotype. This demonstrates Target-AID is a promising genetic tool for Lactobacilli and can accelerate both applied and fundamental research. KEY POINTS: • Efficient and multiplexable cytosine base editing established in Lactobacilli. • Edited Lactobacillus reducing imidazole propionate associated with the risk of type 2 diabetes. • Transient knock-out and dissection of an essential gene function.
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Expanding the target range of base editing in plants without loss of efficiency by blocking RNA-silencing.
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