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Title: Hypercompact CRISPR–Cas12j2 (CasΦ) enables genome editing, gene activation, and epigenome editing in plants
Award ID(s):
1758745 2029889
Author(s) / Creator(s):
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Date Published:
Journal Name:
Plant Communications
Page Range / eLocation ID:
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Summary

    Hornworts are crucial to understand the phylogeny of early land plants. The emergence of ‘reverse’ U‐to‐C RNA editing accompanying the widespread C‐to‐U RNA editing in plant chloroplasts and mitochondria may be a molecular synapomorphy of a hornwort–tracheophyte clade. C‐to‐U RNA editing is well understood after identification of many editing factors in models likeArabidopsis thalianaandPhyscomitrella patens, but there is no plant model yet to investigate U‐to‐C RNA editing. The hornwortAnthoceros agrestisis now emerging as such a model system.

    We report on the assembly and analyses of theA. agrestischloroplast and mitochondrial genomes, their transcriptomes and editomes, and a large nuclear gene family encoding pentatricopeptide repeat (PPR) proteins likely acting as RNA editing factors.

    Both organelles inA. agrestisfeature high amounts of RNA editing, with altogether > 1100 sites of C‐to‐U and 1300 sites of U‐to‐C editing. The nuclear genome reveals > 1400 genes for PPR proteins with variable carboxyterminal DYW domains.

    We observe significant variants of the ‘classic’ DYW domain, in the meantime confirmed as the cytidine deaminase for C‐to‐U editing, and discuss the first attractive candidates for reverse editing factors given their excellent matches to U‐to‐C editing targets according to the PPR‐RNA binding code.

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  2. Abstract

    Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into theE. coligenome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.

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