Frontiers in Plant Science, Journal Year: 2024, Volume and Issue: 15
Published: Sept. 10, 2024
Because virus vectors can spread systemically autonomously, they are powerful vehicles with which to deliver genome-editing tools into plant cells. Indeed, a vector based on positive-strand RNA virus, potato X (PVX), harboring SpCas9 and its single guide (sgRNA), achieved targeted mutagenesis in inoculated leaves of
Language: Английский
Biotechnology Advances, Journal Year: 2024, Volume and Issue: 74, P. 108382 - 108382
Published: May 25, 2024
Language: Английский
Citations
14
Advanced Science, Journal Year: 2024, Volume and Issue: 11(19)
Published: Feb. 26, 2024
CRISPR-based gene therapies are making remarkable strides toward the clinic. But large size of most widely used Cas endonucleases including Cas9 and Cas12a restricts their efficient delivery by adeno-associated virus (AAV) for in vivo editing. Being exceptionally small, recently engineered type V-F CRISPR-Cas12f1 systems can overcome cargo packaging bottleneck present as strong candidates therapeutic applications. In this study, pairwise editing efficiencies different Cas12f1/sgRNA scaffold combinations systemically screened optimized, CasMINI_v3.1/ge4.1 system is identified being able to significantly boost activity. Moreover, packaged into single AAV vectors delivered via subretinal injection, achieves remarkably high efficiencies, over 70% transduced retinal cells. Further, efficacy Cas12f1 system-based therapy treat retinitis pigmentosa Rho
Language: Английский
Citations
7
ACS Chemical Biology, Journal Year: 2024, Volume and Issue: 19(7), P. 1399 - 1408
Published: June 20, 2024
The therapeutic application of CRISPR-based gene editing technology is hindered by the delivery challenges large Cas nucleases. emergence miniature tools derived from type V CRISPR systems and their ancestor TnpB nucleases presents promising solutions to counter these obstacles. Notably, CRISPR-Cas12f -Cas12n exhibit not only a concise size but also remarkable precision in targeted editing, thereby underscoring potential as supreme tools. Although both are considered intermediates evolution mature Cas12 effectors, they distinct biochemical structural characteristics, demonstrating diversity complexity TnpB's evolutionary outcomes. diverse branches indicate existence numerous unexplored compact nature, mining development which could potentially revolutionize manipulation techniques pave way for innovative applications therapy. In this Account, we summarize recent advances our group with research Cas12f Cas12n genome systems, including identification, characterization, engineering improving efficiency. Additionally, discuss process ancestral nuclease growing into various giving insight discovery novel systems.
Language: Английский
Citations
6
Molecular Therapy, Journal Year: 2024, Volume and Issue: 32(4), P. 910 - 919
Published: Feb. 12, 2024
Language: Английский
Citations
4
Chinese Journal of Chemistry, Journal Year: 2025, Volume and Issue: unknown
Published: March 18, 2025
Comprehensive Summary Cas12f possesses both cis ‐ and trans ‐cleavage activities, with the former being extensively studied for its application in genome editing, while latter remains less explored, particularly diagnostic purposes, is mostly focused on Un1Cas12f1. In this study, we conducted a comprehensive comparison of activities four characterized proteins, demonstrating that all exhibit ‐DNase activity triggered by double‐stranded DNA (dsDNA), single‐stranded (ssDNA), RNA (ssRNA). Additionally, identified distinct base preferences substrates among these proteins. Our further investigation into revealed intricate relationship between under various conditions. study provides multifaceted characterization features nucleases, offering new avenues deeper comprehension mechanisms underlying Cas12f's functionality.
Language: Английский
Citations
0
Biotechnology and Bioengineering, Journal Year: 2025, Volume and Issue: unknown
Published: March 19, 2025
ABSTRACT Miniature CRISPR/Cas systems possess delivery advantages for gene therapy. The type V‐F Cas12f1 from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been engineered by several studies as genome editing tools through protein single guide RNA (sgRNA) engineering. However, a comparative evaluation of activation efficiencies mediated different AsCas12f1 variants sgRNA scaffolds lacking. This study tested combinations four six their transcription efficiencies. variant AsCas12f1‐HKRA performed the best in when paired with sgRNA‐en_v2.1 scaffold. Furthermore, we validated super miniature activator fusing small domain to AsCas12f1‐HKRA. Our findings recommend using applications.
Language: Английский
Citations
0
ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(7), P. 2115 - 2127
Published: June 28, 2024
Cas12f nucleases are one of the most compact genome editors, exhibiting promising potential for in vivo therapeutic applications. However, availability active editors remains relatively limited field. Here, we report characterization and engineering a novel miniature endonuclease from Eubacterium siraeum (EsCas12f1, 433 amino acids). We elucidate specific Protospacer Adjacent Motifs preference detailed biochemical properties DNA targeting cleavage. By employing rational design strategies, systematically optimize guide RNA EsCas12f1, converting initially ineffective CRISPR-EsCas12f1 system into an efficient bacterial editor. Furthermore, demonstrate capacity EsCas12f1 vitro nucleic-acid diagnostics. In summary, our results enrich CRISPR-Cas toolbox pave way application both editing
Language: Английский
Citations
3
MedComm, Journal Year: 2024, Volume and Issue: 5(7)
Published: July 1, 2024
The development of gene editing tools has been a significant area research in the life sciences for nearly 30 years. These have widely utilized disease detection and mechanism research. In new century, they shown potential addressing various scientific challenges saving lives through therapies, particularly combating cardiovascular (CVD). rapid advancement therapies provided optimism CVD patients. progress therapy CVDs is comprehensive reflection practical implementation technology both clinical basic settings, as well steady treatment CVDs. This article provides an overview commonly DNA-targeted developed thus far, with specific focus on application these tools, clustered regularly interspaced short palindromic repeat/CRISPR-associated genes (Cas) (CRISPR/Cas) system, therapy. It also delves into limitations current while summarizing ongoing trials related to CVD. aim facilitate further exploration by relevant researchers successful applications field
Language: Английский
Citations
2
Communications Biology, Journal Year: 2024, Volume and Issue: 7(1)
Published: Oct. 12, 2024
Cas12 and Cas13 are extensively utilized in molecular diagnostics for their trans-cleavage activities, yet activation characteristics remain partially understood. Here, we conduct an in-depth investigation of Cas12a, Cas12f1, Cas13a, uncovering the trans-DNase trans-RNase activities with noncanonical activators. Our findings reveal that DNA can serve as a direct target CRISPR-Cas13a, markedly increasing detection sensitivity single-base mismatches. Moreover, Cas12a Cas13a be activated by diverse RNA:DNA RNA:RNA duplexes, respectively, indicating presence stem–loop structures crRNAs is not essential activation. Notably, unlike exhibits intrinsic RNase activity independently Leveraging these insights, have improved accuracy dual-gene approach employs CRISPR-Cas12f1 systems. research advances understanding contributing to field CRISPR-based diagnostics. Uncovering improves enhances single base mismatches through newly discovered mechanisms
Language: Английский
Citations
2
Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(22), P. 14030 - 14042
Published: Nov. 12, 2024
Abstract Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate molecular details underlying AsCas12f1-mediated cleavages. We find following protospacer unwinding non-target strand (NTS) nicking, AsCas12f1 surprisingly carries out bidirectional exonucleolytic cleavage from nick. Subsequently, extended to out-of-protospacer region, gradually digests unwound beyond Eventually, single endonucleolytic target-strand at 3 nt downstream readily dissociates ternary AsCas12f1-sgRNA–DNA complex adjacent motif-distal end, leaving staggered double-strand break. The coupling between both promoted by Mg2+. Kinetic analysis on engineered AsCas12f1-v5.1 variant identifies only accelerated step NTS trimming within sequential cleavage. Our findings provide dynamic view catalyzing unwinding-coupled nucleolytic help with practical improvements Cas12f-based genome editing tools.
Language: Английский
Citations
1