RNA Guided Technologies: siRNA, miRNA & CRISPRi

Question 1

PTGS

Answer 1

post transcriptional gene silencing; phenomenon first identified in plants that has also been shown to occur in animals, although PTGS was initially described as an endogenous method for viral defense and trasposon silencing, it has now emerged as an exciting new research tool, RNA interference

Question 2

RNA interference (RNAi)

Answer 2

a phenomenon where a RNA introduced to a cell ultimately causes the degradation of the complementary cellular mRNA, and leads to the knockdown of gene activity

Question 3

siRNA

Answer 3

short (or small) interfering RNA’ this is a short 21-23 nt RNA duplex involved in eliciting the RNAi response in mammalian cells

Question 4

miRNA

Answer 4

evolutionarily conserved, noncoding, RNA originating from longer transcripts characterized by imperfect hairpin structures, miRNAs are 19-23 nt RNAs processed from pre-miRNA precursors

Question 5

RISC

Answer 5

RNA-induced silencing complex; this is the proposed multi-protein complex that acts to bring the antisense strand of the siRNA and the cellular mRNA together; and endonucleolytic activity associated with the RISC cleaves the mRNA, which is then released and degraded

Question 6

Uses of RNAi

Answer 6

determine gene function
study pathways
generate knockdown or knockouts
identify and validate targets
specifically silence disease-causing genes
target human pathogens
pharmacogenomics

Question 7

Guidelines for siRNA design

Answer 7

AA(N10)TT or NA(N21) target DNA sequences
30-70% GC content
avoid stretches of >4 T’s or A’s
avoid introns, untranslated regions, and sequence within 100 bps of the start
select 2-4 siena target sites

Question 8

shRNA

Answer 8

clone plasmid into cell with the siRNAs that you want

Question 9

Ways to generate siRNAs

Answer 9

chemical synthesis
in vitro transciption
digestion of long dsRNAs in vitro by RNase III or Dicer

Question 10

Ways to deliver siRNAs

Answer 10

direct injection
conjugated siRNAs targeted to specific cell receptors
lipoplexes
viral vectors

Question 11

Controls for siRNA

Answer 11

no treatment
negative (non-specific or scrambled)
positive (assay target mRNA levels to monitor transfection efficiency and RNAi induction)
titration of the siRNA (find the best concentration)
monitoring of protein levels and mRNA levels
monitor off target effects and antiviral responses

Question 12

Steps for successful siRNA experiment

Answer 12

use 2-4 siRNAs per gene target
lowest concentration possible
avoid RNases
maintain healthy cultures
avoid antibiotics
optimize transfection conditions
use characterized negative controls
use positive control
monitor mRNA and protein levels

Question 13

Biological barriers of RNAi therapies

Answer 13

degradation
penetrance into specific tissues
intracellular delivery and trafficking

Question 14

Toxicities of RNAi

Answer 14

immune response
carrier toxicity
off-target effects

Question 15

tissue specificity for RNAi therapies

Answer 15

healthy normal cell vs cancer

Question 16

Clinical challenges with RNAi therapies

Answer 16

biological barriers
toxicities of RNAi
tissue specificity
monitoring delivery and expression

Question 17

miRNA facts

Answer 17

discovered in 1993
mammalian genome has 200-1000 that regulate 1/3 of all genes
doesn’t cause cleavage, but prevents transcription

Question 18

CRISPR

Answer 18

clustered, regular interspaced, short palindromic repeats
defense system against viral attack in bacteria and archaea
memory chip of viruses

Question 19

How CRISPR works

Answer 19

virus invades
CRISPR RNA binds viral genome through complementary base pairing
the Cas9 endonuclease cuts the viral DNA, halting the attack

Question 20

CRISPRi

Answer 20

uses a sgRNA and catalytically dead Cas9

the Cas9 can’t break the dsDNA and just sits there blocking transcription