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The CRISPR technologies enabling precise genome manipulation are valuable for the gene function study and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM), such as NGG, TTN, etc., for Cas protein recognition restricts the targetable genomic loci in applications. Very lately, Cas9-NG which recognizes a minimal NG PAM was reported to expand the targeting space in genome editing in human cells, but little is known about its applications in plants. Here, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice, and revealed that Cas9-NG edits all NGG, NGA, NGT and NGC sites with impaired activities, and gene-edited plants were dominated by mono-allelic mutations. The Cas9-NG-engineered base editors were further developed and used to generate OsBZR1 gain-of-function plants, which other available Cas9-engineered base editors are not able to. Moreover, Cas9-NG-based transcriptional activator efficiently up-regulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, NCG apart from NG PAM. All these findings show that Cas9-NG greatly expands the targeting scope of genome-editing tools and imply great potentials for targeted genome editing, base editing and genome regulation in plants.
This article was published in the following journal.
Name: Molecular plant
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Genetic engineering techniques that involve DNA REPAIR mechanisms for incorporating site-specific modifications into a cell's genome.
The integration of exogenous DNA into the genome of an organism at sites where its expression can be suitably controlled. This integration occurs as a result of homologous recombination.
An APOBEC deaminase catalytic subunit of the apolipoprotein B (APOB) MESSENGER RNA (mRNA) editing enzyme complex that is involved in post-transcriptional editing of a CAA codon for GLYCINE to a UAA STOP CODON in the ApoB mRNA. It also functions in CGA (ARGININE) to UGA STOP CODON editing of NEUROFIBROMIN 1 mRNA and EPIGENETIC PROCESSES.
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A process that changes the nucleotide sequence of mRNA from that of the DNA template encoding it. Some major classes of RNA editing are as follows: 1, the conversion of cytosine to uracil in mRNA; 2, the addition of variable number of guanines at pre-determined sites; and 3, the addition and deletion of uracils, templated by guide-RNAs (RNA, GUIDE).
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