Genomic Editing

Question 1

What is genomic editing

Answer 1

A set of techniques which allow for the identification, manipulation and
multiplication of specific genes in living organisms.

Question 2

Objectives of genomic editing

Answer 2

to create transgenic plants/animals

to study function & structure of genes

to produce useful products

to treat rare genetic diseases by investigating genome modification

Question 3

RNAi

Answer 3

RNAs that inhibit gene transcription

work at post-transcriptional level

Question 4

Normal functions of RNAi in cells

Answer 4

Surveillance against viruses

turn on/off genes during development

Question 5

3 types of RNAi

Answer 5

shRNA - short hairpin RNAs

miRNA - micro RNAs

siRNA - small interferring RNAs

Question 6

miRNA path

Answer 6

miRNA gene transcribed to pri-miRNA the cleaved by DROSHA to pre-miRNA

exported from nucleus
DICER cleaves to miRNA
loaded onto RISC
passenger strand discarded

not fully complementary to target RNA
bind 3’UTR

= Translation repression
= mRNA cleavage
= mRNA degradation

Question 7

siRNA path

Answer 7

Starts off double-stranded
cleaved by DICER into siRNAs
loaded onto RISC
passenger strand is cleaved

fully complementary to target RNA
=mRNA cleavage

Question 8

How targets does siRNA have ?

miRNA have ?

Answer 8

siRNA - one target

miRNA - many targets

Question 9

miRNA invovled in many processes

Answer 9

stress response 
neurodevelopment
extinction of fearful memories 
regulate circadian rhythms 
Targets for Na+2 valporate, lithium, cocaine, fluoxetine

Question 10

miRNA biomarkers

miR-1202
miR-135
miR-16
miR-182
miR-219

Answer 10

1202 - predicts response to anti-depressants

135 & 16 - mediate response to anti-depressants

182 - inhibits fear learning

219 - promotes schizophrenia like behaviors

Question 11

Uses for siRNA ?

Uses for miRNA ?

Answer 11

siRNA - therapeutic agent

miRNA - therpeutic agent, drug target, diagnostic and biomarker tool

Question 12

siRNA & shRNA delivery

Answer 12

siRNA - non-viral

shRNA - viral

Question 13

siRNA non-viral routes

Answer 13

cholestrol - bound to 3’ terminus of passenger strand
transported in blood bound albumin
binds the lowdensity lipo protein receptors
endocytosis

liposomes - engulfed by liposomes called stable nucleic acid lipid particles SNALPs
increases transport in blood after intravenous injection
endocytosis/ fuse directly with membrane

Antibody fragments - By combining the nucleic acid–bindingproperties of protamine (small, arginine rich,
nuclear proteins) and the specificity
of an antibody
an antibody fragment protamine
fusion protein can noncovalently bind siRNAs and deliver siRNAs, probably by endocytosis,
specifically to cells that express the
surface receptor recognized by the
antibody

Question 14

CRISPR stands for

Answer 14

Clustered Regularly Interspaced short pallindromic repeats

Question 15

CRISPR components

Answer 15

CAS 9 - DNA cutting enzyme

guide RNA

Question 16

CAS9 cuts DNA where ?

Answer 16

before PAM

Question 17

The guide RNA is complementary to ?

Answer 17

sequence opposite target gene

Question 18

Cutting results in either ?

Answer 18

Non- homologus end joining

homology directed repair (introducing foreign DNA)

Question 19

miRNAs packaged and sent to extracellular environment in 3 ways

Answer 19

1 enclosed in membraneous vesicles including exosomes, shedding vesicles and apoptotic bodies

2 associated with lipoprotein e.g HDL

3 asoociated with RNA-binding proteins
AGO2 -argonaute 2
NPM1 - nucleophosmin 1