It is shown that nuclear‐expressed sgRNA can be delivered into chloroplasts by fusion with a viroid RNA, as one possible approach for RNA‐guided engineering of the ptDNA without direct chloroplast genome transformation.
Abstract
ABSTRACT Our goal is to develop RNA‐guided engineering of the chloroplast genome using the CRISPR/Cas9 system. We designed chloroplast minigenes to obtain properly sized single guide RNAs (sgRNAs) in tobacco chloroplasts. The sgRNA 5′ end is defined by transcription from an rRNA operon promoter, and its 3′ end by processing a downstream tRNA (trnG) or a hepatitis delta virus (HDV) ribozyme. Cas9 is expressed from a nuclear gene and is targeted to chloroplasts by fusion to a transit peptide. Cas9 incorporated the sgRNA and introduced double‐strand breaks in the plastid DNA (ptDNA). We report here that the double‐strand DNA break in the ndhA and rpoC1 genes was repaired by microhomology‐mediated end joining (MMEJ), resulting in deletions in the ptDNA. We further showed that nuclear‐expressed sgRNA can be delivered into chloroplasts by fusion with a viroid RNA, as one possible approach for RNA‐guided engineering of the ptDNA without direct chloroplast genome transformation. These results are the first step of RNA‐guided editing of the chloroplast genome in any crop.
This study establishes a streamlined and highly efficient framework for precise insertional mutagenesis and double-mutant generation in Ulva, thereby expanding the genetic engineering toolkit for this macroalga.
K. Ichihara, Chikako Nagasato, T. Yamazaki et al.· Phycology Journal· 0 citations
An RNA-guided bridge recombinase system is engineered through rational mutagenesis and AI-assisted directed evolution, enabling programmable chromosomal rearrangements in both plant and mammalian cells and achieving up to a 29.8-fold increase in activity.
Rui Gao, Jingjing Wei, Chao Sun et al.· Trends in Biotechnology· 0 citations
Tandem Interspaced Guide RNA (TIGR)–TIGR-associated (Tas) systems are a newly discovered family of ultracompact, modular RNA-guided DNA-targeting proteins that function without a protospacer adjacent motif (PAM) requirement. Their utility as genome engineering tools in microbes remains unexplored. Here, we report the first functional implementation of TIGR-Tas in Saccharomyces cerevisiae for genome engineering. We show that TasR from Parcubacteria (ParTasR) can be programmed by user-defined tigRNAs to generate targeted DNA double-strand breaks at yeast endogenous loci. By co-delivering ParTasR with customized tigRNAs and donor templates, we achieved precise gene fragment deletion and targeted codon substitutions at multiple genomic loci. The multiplex genome engineering capability of this TIGR-Tas system was demonstrated through high-efficiency multiplex gene disruption and chromosomal assembly of a lycopene biosynthesis pathway while inactivating an endogenous gene. This work establishes TIGR-Tas as a valuable addition to the yeast genome engineering toolbox, particularly for applications requiring PAM-independent targeting or compact delivery.
TnpB proteins are among the most abundant genes encoded in bacterial and archaeal genomes, yet their function in the transposon life cycle remained unclear for decades. Recognition that TnpB is the likely evolutionary ancestor of type V CRISPR-Cas12 effector nucleases, followed by experimental demonstration that TnpB is itself a compact RNA-guided DNA endonuclease, has transformed these previously regarded accessory transposon proteins into a promising frontier in genome engineering. At roughly 350-410 amino acids, TnpB is less than half the size of Cas12a and approximately one-third the size of Cas9, making it well suited to delivery vehicles with constrained cargo capacity, such as adeno-associated virus (AAV). This review discusses recent progress in TnpB biology and technology. It first describes the biochemical and structural basis of RNA-guided DNA cleavage by TnpB and its role in transposon homing. It then summarises comparative genomic analyses that reveal the diversity of TnpB, repeated independent evolutionary transitions from TnpB to Cas12, and recurrent exaptation of TnpB for cellular functions unrelated to transposition. The review also considers how mining natural TnpB diversity and high-throughput protein engineering have produced compact editors with activity and specificity approaching established CRISPR-Cas tools in selected contexts, including early demonstrations of TnpB-mediated editing in animals and crop plants. It concludes by outlining the principal challenges, including transposon-associated motif (TAM) restriction, off-target activity, delivery and mechanistic understanding, that must be addressed before the therapeutic and agricultural potential of TnpB-derived technologies can be fully realised.
Sanchit Pal Singh, Shruti Gupta, Rohit Solanki et al.· Journal of Advances in Biolo...· 0 citations
This chapter describes the design of cgRNAs and provides detailed protocols for their in vivo characterization in E. coli, and shows how cgRNAs can be integrated into endogenous gene circuits to achieve sophisticated and logical regulation of gene expression.
Dongwon Park, Woosub Shin, Hansol Kang et al.· Methods in molecular biology· 0 citations
Abstract RNA-guided obligate mobile element guided activity systems derived from transposable elements have emerged as compact genome-editing tools that may replace clustered regularly interspaced short palindromic repeats platforms. We established a dual-mode genome regulation platform using ISDge10 TnpB effectors and engineered ωRNAs, in which modulation of the ωRNA guide length enables switching between programmable transcriptional activation and genome editing. TnpB programmed with a 10-nt guide region of the ωRNA engages target DNA without inducing double-strand breaks. Fusion of transcriptional activators with Sso7d (DNA-binding protein from Sulfolobus solfataricus) enables specific transcriptional upregulation across endogenous loci. Restoring the ωRNA guide length to 20 nt triggers DNA cleavage, thereby supporting homology-directed repair-mediated sequence correction. A catalytically inactivated TnpB-based adenine base editor enabled A-to-G base conversion at genomic targets. TnpB shows strict ωRNA-dependent mismatch sensitivity with low off-target effects, suggesting its potential as a high-fidelity genome regulation platform. Compact ISDge10 TnpB facilitates co-packaging of effector and ωRNA in a single adeno-associated virus vector and co-expression of large functional domains. Thus, this study expands RNA-guided genome-editing capabilities.
Yeounsun Oh, Se‑Been Jeon, Lee Wha Gwon et al.· Nucleic Acids Research· 0 citations
