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Found: 26 149
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Exonuclease-enhanced prime editors.
Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.
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CRISPR-Mediated Silkworm: The Oncoming Agricultural Revolutions and a Rising Model Organism.
The silkworm (Bombyx mori) is essential to sericulture and is also becoming a key model organism in genomics and agriculture. For decades, genetic studies of the silkworm were limited by inefficient and inflexible genome tools. CRISPR genome editing allows precise and scalable alterations to genes regulating development, physiology, and industrial traits. This review summarizes silkworm genome-editing breakthroughs, highlighting CRISPR's evolution from simple gene knockouts to large-scale genome-wide screening. We highlight how these advancements contribute to disease resistance, higher yields, and the development of new silk-based materials, as well as how they influence the development and growth rate of the sericulture. The creation of high-quality reference genomes, pangenomes, and genome-wide screening systems has made the silkworm a major model for integrating multiple biological datasets and approaches, such as genomic, transcriptomic, and proteomic. By considering the unique biological characteristics of the silkworm, this provides new insights for research on silk biology, piRNA synthetic biology, and hormonal signaling regulation. Finally, we examine new areas at the intersection of CRISPR, pangenomics, and artificial intelligence (AI) and suggest future paths for molecular breeding, pest control, and synthetic biology. Moreover, AI-assisted prediction of CRISPR outcomes is utilized to inform the design of targeted trait modifications, representing an approach to enhancing biomanufacturing efficiency and eco-friendly silk production. Together, these advances have made the silkworm a flexible genetic platform and an important part of sustainable agriculture and biomanufacturing.
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Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity.
Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.
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Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates.
RNA modification in the form of N6-methyladenosine (m6A) regulates nearly all the post-transcriptional processes. The asymmetric m6A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m6A modifications. Here we report the development of 'm6A editing', a powerful approach that enables m6A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m6A methyltransferase that can be programmed with a guide RNA. The resultant m6A 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m6A 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m6A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.
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CRISPR/Cas12a Mediated Genome Editing To Introduce Amino Acid Substitutions into the Mechanosensitive Channel MscCG of Corynebacterium glutamicum.
Against the background of a growing demand for the implementation of environmentally friendly production processes, microorganisms are engineered for the large-scale biosynthesis of chemicals, fuels, or food and feed additives from sustainable resources. Since strain development is expensive and time-consuming, continuous improvement of molecular tools for the genetic modification of the microbial production hosts is absolutely vital. Recently, the CRISPR/Cas12a technology for the engineering of Corynebacterium glutamicum as an important platform organism for industrial amino acid production has been introduced. Here, this system was advanced by designing an easy-to-construct crRNA delivery vector using simple oligonucleotides. In combination with a C. glutamicum strain engineered for the chromosomal expression of the β-galactosidase-encoding lacZ gene, this new plasmid was used to investigate CRISPR/Cas12a targeting and editing at various positions relative to the PAM site. Finally, we used this system to perform codon saturation mutagenesis at critical positions in the mechanosensitive channel MscCG to identify new gain-of-function mutations for increased l-glutamate export. The mutations obtained can be explained by particular demands of the channel on its immediate lipid environment to allow l-glutamate efflux.
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Gene editing of MPS I human fibroblasts by co-delivery of a CRISPR/Cas9 plasmid and a donor oligonucleotide using nanoemulsions as nonviral carriers.
Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by the deficiency of alpha-L-iduronidase (IDUA). This study shows the use of nanoemulsions co-complexed with the plasmid of CRISPR/Cas9 system and a donor oligonucleotide aiming at MPS I gene editing in vitro. Nanoemulsions composed of MCT, DOPE, DOTAP, DSPE-PEG, and water were prepared by high-pressure homogenization. The DNA was complexed by adsorption (NA) or encapsulation (NE) of preformed DNA/DOTAP complexes with nanoemulsions at +4/-1 charge ratio. The incubation in pure DMEM or supplemented with serum showed that the complexation with DNA was stable after 1 h of incubation, but the complexes tended to release the adsorbed DNA after 24 h of incubation, while the encapsulated DNA remained complexed in the oil core of the nanoemulsions even 48 h after incubation with DMEM. The treatment of MPS I patient's fibroblasts homozygous for the p.Trp402∗ mutation led to a significant increase in IDUA activity at 2, 15, and 30 days when compared to MPS I untreated fibroblasts. Flow cytometry and confocal microscopy demonstrated that there was a reduction in the area of lysosomes to values similar to normal, an indicator of correction of the cellular phenotype. These results show that the nanoemulsions co-complexed with the CRISPR/Cas9 system and a donor oligonucleotide could effectively transfect MPS I p.Trp402∗ patient's fibroblasts, as well as enable the production of IDUA, and represent a potential new treatment option for MPS I.
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Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with wide-ranging genome-editing efficiencies that differ in PAM-binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome-editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome-editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.
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QBEmax is a sequence-permuted and internally protected base editor.
Cytosine base editors (CBEs) show promise for multiplex gene knockout applications, but impure edits, indels and off-targets still frequently occur. We describe here QBEmax, which exhibits high efficiency, low indel and off-targets and high product purity with up to 99.8% of edits comprised of C-to-T. Through molecular dynamic modeling, QBEmax presents as a compact and stable base editor that shields protected bases from undesired repair processes.
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Addition of the T5 exonuclease increases the prime editing efficiency in plants.
Prime editing (PE) is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates, but is limited by low editing efficiency. We previously fused the M-MLV reverse transcriptase to the Cas9 nickase, generating the PE2 (v1) system, but the editing efficiency of this system is still low. Here we develop different versions of PE2 by adding the 5'-to-3' exonuclease at different positions of the nCas9-M-MLV RT fusion protein. PE2 (v2), in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions, deletions, and insertions at several genomic sites by 1.7- to 2.9-fold in plant cells compared to PE2 (v1). The improved editing efficiency of PE2 (v2) is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1, PE-P2, and PPE systems. Using PE2 (v2), we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites. The PE efficiency is further improved by combining PE2 (v2) and dual-pegRNAs. Taken together, the increased genome editing efficiency of PE2 (v2) developed in this study may enhance the applications of PE in plants.
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Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9.
Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules, and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.
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