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From IscB to Cas9: Engineering and advances in the next generation of miniature gene editing tools.
The CRISPR-Cas system, distinguished by its inherent modularity and broad programmability, has catalyzed a paradigm shift in genome engineering due to its unprecedented accuracy, specificity, and on-target efficiency, now serving as the cornerstone of modern genome manipulation. The efficient delivery of gene editing tools remains a major technical hurdle to clinical application, primarily due to the lack of compact editors. The recent identification of the transposon-associated nuclease IscB as an evolutionary ancestor of Cas9 has provided important insights into the molecular evolution of the CRISPR-Cas9 system. Notably, IscB is a highly compact nuclease, approximately one-third the size of Cas9, capable of precise nucleic acid cleavage in eukaryotic cells under the guidance of ωRNA. These features make it a promising candidate for the development of next-generation miniaturized genome editors. However, natural IscB exhibits limited editing performance in eukaryotic systems. This review first outlines the biochemical function of the transposon IscB and briefly traces the evolutionary origin of the Cas9 system. It then describes and compares the structural characteristics and cleavage mechanisms of OgeuIscB and Cas9. Subsequent sections summarize various engineering strategies for current IscB systems, including the development of base editors and recent advances in their application. Finally, the limitations of existing systems are discussed, and potential directions for future optimization are proposed, aiming to provide new insights and facilitate the advancement of IscB-based miniaturized editors.
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The engineered single guide RNA structure as a biomarker for gene-editing reagent exposure.
CRISPR arrays and CRISPR-associated (Cas) proteins comprise a prevalent adaptive immune system in bacteria and archaea. These systems defend against exogenous parasitic mobile genetic elements. The adaption of single effector CRISPR-Cas systems has massively facilitated gene-editing due to the reprogrammable guide RNA. The guide RNA affords little priming space for conventional PCR-based nucleic acid tests without foreknowledge of the spacer sequence. Further impeding detection of gene-editor exposure, these systems are derived from human microflora and pathogens (Staphylococcus pyogenes, Streptococcus aureus, etc.) that contaminate human patient samples. The single guide RNA-formed from the CRISPR RNA (crRNA) and transactivating RNA (tracrRNA)-harbors a variable tetraloop sequence between the two RNA segments, complicating PCR assays. Identical single effector Cas proteins are used for gene-editing and naturally by bacteria. Antibodies raised against these Cas proteins are unable to distinguish CRISPR-Cas gene-editors from bacterial contaminant. To overcome the high potential for false positives, we have developed a DNA displacement assay to specifically detect gene-editors. We leveraged the single guide RNA structure as an engineered moiety for gene-editor exposure that does not cross-react with bacterial CRISPRs. Our assay has been validated for five common CRISPR systems and functions in complex sample matrices.
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Enhancing heritable genome editing in soybean by optimizing promoter combinations for the LbCas12a system.
The CRISPR-Cas system, adapted from prokaryotic immune mechanisms, enables programmable DNA targeting and editing. Recent advancements include base editors and prime editors, expanding genetic research applications. The LbCas12a (Cpf1) system offers unique advantages, including simplified operation and multiplex editing, yet achieving stable heritable edits in soybean remains challenging due to its complex paleo-tetraploid genome and transformation constraints. Here we optimized promoter combinations for the LbCas12a editing system to maximize both editing efficiency in the initial (E0) generation and the frequency of homozygous or biallelic mutants in the subsequent (E1) generation. We identified prAtHSP70-1 (At5G02500), prAtEF1αA4 (At5G60390), and prGmUbi1 (Glyma.10G251900) as an effective promoter set, and demonstrated that LbCas12a–crRNA accumulation needs to exceed a defined threshold to achieve efficient target cleavage. These findings provide a practical strategy for enhancing heritable genome editing in soybean and may be broadly applicable to other crops.
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Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations.
Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding for Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal editing outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused to the protein or as excipients) and that lipid nanoparticles (LNPs) encapsulating RNPs can be optimized for enhanced RNP stability, delivery efficiency and editing potency. Specifically, after screening for suitable ionizable cationic lipids and by optimizing the concentration of the synthetic lipid DMG-PEG 2000, we show that the encapsulation, via microfluidic mixing, of adenine base editor and prime editor RNPs within LNPs using the ionizable lipid SM102 can result in in vivo editing-efficiency enhancements larger than 300-fold (with respect to the delivery of the naked RNP) without detectable off-target edits. We believe that chemically defined LNP formulations optimized for RNP-encapsulation stability and delivery efficiency will lead to safer genome editing.
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Efficient generation of the mouse model with a defined point mutation through haploid cell-mediated gene editing.
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Anti-CRISPR protein AcrIIA5 can enhance the activity and security of prime editing.
Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the correct edits, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known "inhibitor" Acr protein can unexpectedly acting as an "enhancer" of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity.
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Versatile and efficient genome editing with Neisseria cinerea Cas9.
The CRISPR/Cas9 system is a versatile genome editing platform in biotechnology and therapeutics. However, the requirement of protospacer adjacent motifs (PAMs) limits the genome targeting scope. To expand this repertoire, we revisited and engineered a compact Cas9 orthologue derived from Neisseria cinerea (NcCas9) for efficient genome editing in mammal cells. We demonstrated that NcCas9 generates genome editing at target sites with N4GYAT (Y = T/C) PAM which cannot be recognized by existing Cas9s. By optimizing the NcCas9 architecture and its spacer length, editing efficacy of NcCas9 was further improved in human cells. In addition, the NcCas9-derived Base editors can efficiently generate base conversions. Six anti-CRISPR (Acr) proteins were identified as off-switches for NcCas9. Moreover, NcCas9 successfully generated efficient editing of mouse embryos by microinjection of NcCas9 mRNA and the corresponding sgRNA. Thus, the NcCas9 holds the potential to broaden the CRISPR/Cas9 toolsets for efficient gene modifications and therapeutic applications.
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CRISPR/Cas9-Mediated Mutagenesis of BrLEAFY Delays the Bolting Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).
Chinese cabbage has unintended bolting in early spring due to sudden climate change. In this study, late-bolting Chinese cabbage lines were developed via mutagenesis of the BrLEAFY (BrLFY) gene, a transcription factor that determines floral identity, using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Double-strand break of the target region via gene editing based on nonhomologous end joining (NHEJ) was applied to acquire useful traits in plants. Based on the 'CT001' pseudomolecule, a single guide RNA (sgRNA) was designed and the gene-editing vector was constructed. Agrobacterium-mediated transformation was used to generate a Chinese cabbage line in which the sequence of the BrLFY paralogs was edited. In particular, single base inserted mutations occurred in the BrLFY paralogs of the LFY-7 and LFY-13 lines, and one copy of T-DNA was inserted into the intergenic region. The selected LFY-edited lines displayed continuous vegetative growth and late bolting compared to the control inbred line, 'CT001'. Further, some LFY-edited lines showing late bolting were advanced to the next generation. The T-DNA-free E1LFY-edited lines bolted later than the inbred line, 'CT001'. Overall, CRISPR/Cas9-mediated mutagenesis of the BrLFY gene was found to delay the bolting time. Accordingly, CRISPR/Cas9 is considered an available method for the molecular breeding of crops.
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In Vivo Base Editing Partially Rescues Bone Dysplasia in a Mouse Model of Hutchinson-Gilford Progeria Syndrome.
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder affecting tissues of mesenchymal origin. Most patients harbor a c.1824C>T/p.G608= variant, commonly described as G608G, in exon 11 of LMNA that leads to aberrant splicing and production of the toxic progerin protein. In addition to cardiovascular, dermal, and adipose tissue deterioration, HGPS mouse models also develop progressive bone dysplasia that occurs in patients. Here we characterize the efficacy of in vivo mutation correction with an adenine base editor (ABE) to rescue structural and functional defects in HGPS transgenic murine bone tissue. Treatment of double-copy transgenic osteoblast cultures with a lentiviral-delivered CRISPR-Cas9 ABE achieved nearly 40% gene correction in vitro, resulting in significant reduction of progerin transcripts and protein, in the absence of selective agents. Furthermore, gene correction improved progeroid osteoblasts' capacity to deposit and mineralize extracellular matrix compared to untreated cultures. In vivo, a single intravenous dose of AAV9-delivered ABE corrected the mutation, achieving ~14%, ~22%, ~10% and < 1% correction in bone by six months of age when administered at P3, P14, 1 and 4 months of age, respectively. Partially rescued bone structural and physical parameters were observed in P14-treated mice with concomitant normalization of gene transcriptional programs and intracellular signaling pathways involved in bone remodeling. This work demonstrates in vivo delivery of a locus-specific DNA base editor to bone tissue, delineates the timing of treatment required for maximum efficacy, and suggests that this system might be tailored for application to other monogenic bone disorders.
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DNA-free genome editing for ZmPLA1 gene via targeting immature embryos in tropical maize.
Doubled haploid (DH) production accelerates the development of homozygous lines in a single generation. In maize, haploids are widely produced by the use of haploid inducer Stock 6, earlier reported in 1959. Three independent studies reported haploid induction in maize which is triggered due to a 4 bp frame-shift mutation in matrilineal (ZmPLA1) gene. The present study was focused on the generation of mutants for ZmPLA1 gene in maize inbred line LM13 through site-directed mutagenesis via CRISPR/Cas9-mediated ribonucleoprotein (RNP) complex method to increase the haploid induction rate. Three single guide RNAs (sgRNAs) for the ZmPLA1 gene locus were used for transforming the 14 days old immature embryos via bombardment. 373 regenerated plants were subjected to mutation detection followed by Sanger's sequencing. Out of three putative mutants identified, one mutant depicted one base pair substitution and one base pair deletion at the target site.
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