Genome Modifications

Transposons

DNA that can mode from place to place in genome, don’t require homology, insert into middle of gene - disruption usually inactivates, carry other genes (selectable marker - antibiotic resistance)

Conservative composite transposons

encode transposase enzyme - cut DNA, have inverted repeats

Transposition steps

Transposase recognize and blunt cuts its inverted repeats, Transposase makes staggered nicks at target, staggeres gaps filled by DNA pol and ligase creating direct repeats flanking transposon

Method for identifying genes responsible for functions

Mutagenize organsim, screen for loss of function, identify what gene mutated - likely responsible for loss of function

Why Composite transposon useful for identifying functions

easy to generate (effective delivery/many mutants to screen), Easy to identify mutant cells (identifiable marker - antibiotic resistance), 1 mutation per cell (likely caused by mutation you generated), easy to identify what you mutated (know sequence of IR - use PCR)

Suicide vector

plasmid that can’t replicate in cell, only mantained in inserted into chromosomes

CRISPR-Cas system

pprokaryotic immune system, degrade foreign nucleic acid, express CRISPR RNA that specifically target invasive nucleic acid, Cas proteins that cut foreign nucleic acid

CRISPR-cas Loci

palindromic repeats, Spacers - non repetitve segment that are fragment of invading DNA (immune memory), cas gene encode protein responsible for activity - different families and subtypes

CRISPR-cas Function

Acquisition of new spacers, Expression and processing of crRNA, CRISPR interference

Acquisition of new spacers

mechanism unknown, nuclease activity of Cas1 to generate short fragments, inserted at side near to leader sequence

CRISPR RNA (crRNA) biogenesis

crRNA transcribed as single transcript than cleaved by Cas proteins, Type I and III need Cas6 protein, Type II use RNaseIII and tracrRNA

Type II CRISPR system

use RNaseIII and trans-activatig crRNA (tracrRNA) for cleavege into crRNA, tracrRNA bp with primary CRISPR transcript - targeted by RNaseIII, mature crRNA don’t have full spacers (truncated), gRNA = crRNA + tracrRNA, PAM recognized on same strand, requires only Cas9 for DNA degradations (endonuclease domain),

CRISPR interference

gRNA generated from crRNA, guide CAs nuclease to target, PAM sequence (2-6bp) 3-4nt downstream of cut site recognized for cleavage, Type V restriction enzymes

Guide RNA (gRNA)

Type 1 and III same as crRNA, Type II is crRNA + tracrRNA, guide Cas nuclease to target

Type I and III CRISPR systems

crRNA cleaved by Cas6, gRNA=crRNA, PAM sequence recognized and bp, conformational change recruit protein that delivers Cas3 for DNA degradation, recruits 6-7 Cas proteins, Type III target mRNA of phages not DNA

Meganuclease

12-40bp recognition site, rarely where we want

Type V restriction enzymes

targeted to sequence by crRNA, could target these enzymes anywhere with engineered crRNA as long as PAM site near

Type II CRISPR-Cas9 system

requires only one cas gene (Cas9), recognize very simple PAM - likley found near target, creates double stranded break, sgRNA, recognize target DNA through ~20nt bp, Cas9 interacts with PAM, Cas9 uses 2 nuclease domains (HNH/RuvC) to cleave dsDNA, creates double stranded break

Synthetic guide RNA (sgRNA)

artificially synthesized crRNA and tracrRNA, targets gene region of interest

CRISPR-Cas9 plasmid

amplify region of genome wish to target with PCR, include in 5’ primer modification to bp with vector overhanges (insert into gRNA), vector codes sgRNA and Cas9, generates ds breaks at target sequence

CRISPR-Cas9 deletion

design 2 sgRNA - each side of region to delete, create 2 ds breaks, allow region to fall out, ends joined by non-homologous end joining

CRISPR-Cas9 insertion

build donor DNA fragment, homology to both sides of ds break, cell uses homologous recombination repair mechanism, donor DNA inserted into ds break

Perspectives on CRISPR-Cas9 genome editing

limitless specificity (newer PAM less), still some off targeted affected area, useful in agriculture (GMO only if added - not removed), moral and ethical question with human gene editing, viral delivery into humans has immune risks, editing germline cells and embryos seems promising