CRISPR, ZFN and TALENs

Question 1

Homologous recombination - pros and cons

Answer 1

exchange of genetic information between between genomic and exogenous DNA via cross over events defined modifications into gene of interest
+: any site on genome, low off target, easy
-: low throughput, labour intensive, low efficiency, requires cells to be dividing, limited species

Question 2

Genome editing (3)

Answer 2

Homologous recombination
endonucleases
CRISPR cas

Question 3

ZFNs

Answer 3

Zinc Finger nucleases (3-6 zinc fingers)
FoKI (cleavage domain) - needs to occur dimerisation (2 adjacent binding events)
Zinc finger (binding domain) - Cys2His2motif - recognizes 3 nucleotides

Question 4

TALENs

Answer 4

Transcription activator-like effector (Xanthomas bacteria) - 15-20
each domain is 33-35 aa long
speciificity defined by variable di-residue (RVDs) responsible for recognition of sequence (NG - T)

Question 5

ZFNs vs TALENs
- precision
-throughput
-efficiency
-off target
- design and production
- money

Answer 5

ZFNs
- medium
-low
-medium-high
-yes
-difficult design and produce
-expensive

TALENs
-high
-medium
-high
-yes
- difficult produce
- quite expensive

Question 6

CRISPR-Cas9 + benefits and limitation
gene knock out workflow

Answer 6

Bacterial:
1- aquisition: small DNA fragment inserted into CRISPR locus (between palindromic sequences) forming new spacer
2- processing (transcription): transcribed into pre-crRNA 39-45nt containing one spacer sequence with stem loop (little hairpin RNA)
3- interference: crRNA recognizes complementary protospacer in viral genome and Cas provide degradation (HNH cuts complementary strand and RuvC cuts non complementary) - DSB

+: easy design and produce, quick, high efficiency and throughput, low cost
-: high frequency of off target effects

1- Design gRNA that matches selected target
2- Select Cas9 variant (usually Streptococcus pyogenes - NGG pam)
3- Delivery (transfection, viral, nanoparticles)
4- validation (PCR, sequencing, off targets)

Question 7

How to decrease off target effects of CRISPR (2) and benefits

Answer 7

Base editing: nickases edit precisely without DSBs or HDR
- Cas9 nickase with a citosine (C - U - A - T) or adenosine (A- I -C - G) and the mismatch repair system copies into complementary strand

Prime editing: directly replace DNA sequence with Cas9 nickase fused to reverse transcriptase, used pegRNA and then endogenous repairs other strand

Benefits: more precise, without cutting, non-dividing cells, minimal off targets

Question 8

Delivery of CRISPR (5)

Answer 8

Plasmid (plasmid with Cas9+gRNA or plasmid with Cas9 and gRNa transfered alongside)

Protein - Cas/gRNA riboprotein complex

mRNA - both as RNA species (pre transcribed Cas9 mRNA)

viral- lentiviral or AAV transduction

lipid nanoparticles or exomes - in vivo delivery of mRNA or protein CRISPR/Cas9

Question 9

validation and detection (2)

Answer 9

PCR - digest with T7 endonuclease - gel- quantify

RE - sequence

Question 10

Design considerations

Answer 10

Which Cas9?
Off target
Knock out: early in coding sequence, common exon
Knock in: close to desired site (<15bp), choice of donor, length of homology arms, selection, prime or base

Question 11

off target detection (5)

Answer 11

Screen predicted off target sites (sanger sequencing)
in vitro cleavage assys
ChIP-seq with dCas9 to identify genome wide binding and targeted NGS
DSB detection/tagging and NGS
WGS