Genome editing techniques allow DNA to be inserted, deleted, modified or replaced in the genome of a living organism. Four families of engineered nucleases have been used for genome editing: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. Each system uses a different mechanism for recognizing and cutting DNA at specific locations to modify genes. The CRISPR/Cas9 system has become widely used due to its ease of design and lower off-target effects compared to other techniques.

1. Genome Editing Techniques
Vikas Verma
PhD Scholar, JNKVV, Jabalpur

2. •Genome editing, or genome engineering, or gene editing, is a type
of genetic engineering in which DNA is inserted, deleted, modified or
replaced in the genome of a living organism.
•Unlike early genetic engineering techniques that randomly inserts
genetic material into a host genome, genome editing targets the
insertions to site specific locations.
What is Genome Editing?

3. •As of 2015 four families of engineered nucleases were
used: meganucleases, zinc finger nucleases (ZFNs), transcription activator-
like effector-based nucleases (TALEN), and the clustered regularly
interspaced short palindromic repeats (CRISPR/Cas9) system.
•Nine genome editors were available as of 2017.
•All three major classes of these enzymes—zinc finger nucleases (ZFNs),
transcription activator-like effector nucleases (TALENs) and engineered
meganucleases—were selected by Nature Methods as the 2011 Method of the
Year.
•The CRISPR-Cas9 system was selected by Science as 2015 Breakthrough of
the Year.
Genome editiors

5. •Meganucleases, discovered in the late 1980s, are enzymes in
the endonuclease family which are characterized by their capacity to
recognize and cut large DNA sequences (from 14 to 40 base pairs).
•The most widespread and best known meganucleases are the proteins in the
LAGLIDADG family, which owe their name to a conserved amino acid
sequence.
•Meganucleases have the benefit of causing less toxicity in cells than
methods such as Zinc finger nuclease (ZFN), likely because of more
stringent DNA sequence recognition.
•One major drawback is the construction of sequence-specific enzymes for
all possible sequences is costly and time consuming, as one is not benefiting
from combinatorial possibilities that methods such as ZFNs and TALEN-
based fusions utilize.
Meganucleases

7. What is ZFN technology?
Zinc fingers were first discovered in the African clawed toad (Xenopus
laevis) in 1985
A class of engineered DNA-binding proteins
Facilitate targated editing of the genome by creating double strand breaks
in the DNA at specified locations
Double strand breaks are importand for site-specific mutagenesis
Stimulate the cell’s natural DNA repair processes i.e, HR and NHEJ
Generate precisely targeted genomic editing resulting in cell lines with
targated gene deletions, integrations, or modifications

8. What are zinc finger nuclease
Highly specific genomic scissor
Consists of two functional domains
• A DNA – binding domain
• A DNA- cleaving domain comprises of nuclease domain of FoK I

9. Diagrammatic representation of ZFN technology
A pair of ZFNs, each with three zinc
fingers binding to target DNA
double strand break
FokI domain

10. Applications of ZFN
•Repairing mutations
•Insertion of gene or DNA fragment at specific site
•Repair or replace aberrant genes
•Disabiling an allele
•Allele editing
•Applications in medical sector
• a) Gene therapy
• b)Treatment of HIV

11. TALENs :Transcription activator-like effector nucleases
TALENs are the restriction enzyme engineered to cut specific
sequences of DNA
They are made by fusing:
DNA-binding domain (TAL effector)
DNA-cleavage domain ( the catalytic domain of RE FoK I)
TALENs can be engineered to bind any desired DNA sequence
to cut at specific locations in DNA

12. TALEN constructs are used in a similar way to designed zinc finger
nucleases
 And have three advantages in targeted mutagenesis:
1. DNA binding specificity is higher
2. off-target effects are lower, and
3. construction of DNA-binding domains is easier
 Based on the maximum theoretical distance between DNA binding
and nuclease activity, TALEN approaches result in the greatest
precision.

13. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are
genetic elements that bacteria use as a kind of acquired immunity to protect
against viruses.
They consist of short sequences that originate from viral genomes and have
been incorporated into the bacterial genome.
Cas (CRISPR associated proteins) process these sequences and cut matching
viral DNA sequences.
By introducing plasmids containing Cas genes and specifically constructed
CRISPRs into eukaryotic cells, the eukaryotic genome can be cut at any desired
position.
Several companies, including Cellectis and Editas have been working to
monetize the CRISPR method while developing gene-specific therapies
CRISPRs

15. Components of CRISPR
1. Proto spacer adjacent motif (PAM)
2. CRISPR RNA (crRNA)
3. Trans activating crRNA (tracr RNA )

17. Nuclease platforms ZFN TALEN CRISPR/Cas9
Source Bacteria, Eukaryotes Eukaryotes Bacteria (Streptococcus sp.)
DNA binding determinant Zinc finger protein
Transcription-activator-like
effector
crRNA/sgRNA
Binding specificity 3 Nucleotides 1 Nucleotide 1:1 Nucleotide pairing
Mutation rate (%) 10 20 20
Target site length (bp) 18–36 24–40 22
Endonuclease Fok I Fok I Cas9
Double-stranded break
pattern
Staggered cut (4–5 nt, 5′
overhang)
Staggered cut
(Heterogeneous overhangs)
Sp Cas9 creates blunt ends;
Cpf1 creates staggered cut
(5′ overhang)
Off-target effects High Low Variable
Ease of design Difficult Moderate Easy
Dimerization required Yes Yes No
Methylation sensitive Yes Yes No
Best suited for
Gene knockout,
Transcriptional regulation
Gene knockout,
Transcriptional regulation
Gene knockout,
Transcriptional regulation,
Base editing
Applications
Human cells, pig, mice,
tobacco, nematode and
zebrafish
Human cells, water flea,
cow and mice
Human cells, wheat, rice,
maize and Drosophila
Comparison of ZFN, TALEN, CRISPR-Cas9 Technologies