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#guide RNA

3 public questions tagged with this topic.

Transgenic Plant -2

CRISPR/Cas9 specificity is provided by:

Specificity of CRISPR Cas9 system is dictated predominantly by guide RNA rather than protein itself, contrasting with zinc finger and TALEN protein-guided recognition. Cas9 from Streptococcus pyogenes contains recognition lobe and nuclease lobe with HNH and RuvC domains, plus PAM interacting domain at C-terminus that scans DNA for 5'-NGG-3' PAM via major groove interactions, required for binding. Spontaneous DNA unwinding initiates RNA-DNA heteroduplex formation with 20 nt spacer of crRNA embedded in single guide RNA fusion with tracrRNA scaffold. Complementarity drives conformational activation: full pairing especially in seed region nucleotides 1-8 proximal to PAM triggers HNH domain rotation cutting target strand complementary, RuvC cutting noncomplementary strand. Mismatches in PAM distal region tolerated leading to off-targets predictable by computational tools. Therefore reprogramming requires only swapping 20 nt spacer chemically synthesized within hours, whereas Cas9 protein unchanged. This RNA-guided programmable nature underlies ease and multiplex ability simply expressing multiple guides, enabling library screening, lineage tracing and epigenome editing fusing dCas9 to effectors where guide directs localization.

Ref: Jinek et al. Science 2012 337:816 Cas9 guide RNA; Nishimasu Cell 2014 156:935 specificity.

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Processing of mRNA, rRNA, tRNA

rRNA chemical modification is guided by

In eukaryotes, precursor ribosomal RNA transcribed as forty-seven S transcript in nucleolus undergoes extensive covalent base modifications essential for ribosome assembly, stability, and decoding accuracy. Modifications include about one hundred 2'-O-methylations and one hundred pseudouridylations guided not by protein enzymes recognizing sequence alone but by small nucleolar RNAs snoRNAs providing sequence complementarity to target site and positioning catalytic proteins fibrillarin for methylation and dyskerin for pseudouridylation. SnoRNAs are intron-encoded, highly abundant within dense fibrillar component of nucleolus where rRNA transcription and early processing occur coordinated with assembly factors.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 6: snoRNA guides rRNA chemical modification; Lodish 9th ed., Nucleolar rRNA processing

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Processing of mRNA, rRNA, tRNA

Guide RNAs are required for RNA editing in

In mitochondria of trypanosomatids including Trypanosoma brucei, many protein-coding genes exist as cryptogenes requiring extensive uridine insertion and deletion editing guided by small noncoding guide RNAs encoded in kinetoplast DNA. Guide RNAs form short anchor duplex with pre-messenger RNA upstream of editing sites, specifying positions via base pairing where editosome complex containing endonuclease, 3' terminal uridylyltransferase TUTase adding uridines, and RNA ligase executes insertions. Editing can add hundreds of uridines and delete dozens, dramatically remodeling open reading frames to generate functional cytochrome oxidase subunits and other mitochondrial proteins essential for respiration.

Ref: Berg et al., Biochemistry, 9th ed., Chapter 28: Guide RNAs required for editing mitochondria of trypanosomes; Cell, Trypanosome editosome structure

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