Engineering an optimized hypercompact CRISPR/Cas12j‐8 system for efficient genome editing in plants

Plant Biotechnology Journal, Год журнала: 2025, Номер unknown

Опубликована: Янв. 12, 2025

Summary The Cas12j‐8 nuclease, derived from the type V CRISPR system, is approximately half size of Cas9 and recognizes a 5′‐TTN‐3′ protospacer adjacent motif sequence, thus potentially having broad application in genome editing for crop improvement. However, its efficiency remains low plants. In this study, we rationally engineered both crRNA nuclease. markedly improved When combined, they exhibited robust activity soybean rice, enabling target sites that were previously uneditable. Notably, certain sequences, was comparable to SpCas9 when targeting identical it outperformed Cas12j‐2 variant, nCas12j‐2, across all tested targets. Additionally, developed cytosine base editors based on Cas12j‐8, demonstrating an average increase 5.36‐ 6.85‐fold base‐editing (C T) compared with unengineered system plants, no insertions or deletions (indels) observed. Collectively, these findings indicate hypercompact CRISPR/Cas12j‐8 serves as efficient tool mediated by nuclease cleavage

Язык: Английский

Discovery of widespread activating mutations in a compact RNA-guided endonuclease

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Фев. 12, 2025

TnpB is an evolutionarily diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship Cas12s, holds significant potential for genome editing mechanistic exploration. However, most TnpBs lack robust gene-editing activity, unbiased profiling mutational effects on activity has not been experimentally explored. Here, we mapped comprehensive sequence-function landscapes a ribonucleoprotein discovered many activating mutations in both the protein RNA. Single-position changes RNA outperform existing variants, highlighting utility systematic scaffold mutagenesis. Leveraging landscape protein, identified enhanced variants from combinatorial library mutations. These increased human cells N. benthamiana by over two-fold fifty-fold relative wild-type TnpB, respectively. In total, this study highlights unknown elements critical regulation endonuclease RNA, reveals surprising amount latent accessible through mutation.

Язык: Английский

Progress on medicinal plant regeneration and the road ahead

Medicinal Plant Biology, Год журнала: 2020, Номер 0(0), С. 1 - 16

Опубликована: Янв. 1, 2020

Язык: Английский