What is Gene Editing? HDR for Genome Editing. Zinc Finger Nucleases increase the rate of recombination. TALE Proteins.

1. Gene Editing

2. What is gene editing?
• Making precise alterations to the genome
• Removal of DNA
• Insertion of DNA
• Base pair alterations of the DNA

3. How do we edit the genome?
• At the heart of genome editing is the ability to make a precise cut at a
specific sequence within the genome
Creation of frameshifts
Insertion of DNA
Alterations to the DNA sequence
Removal of DNA
Requires the activity of an endonuclease

4. HDR for genome editing
An Acad Bras Cienc. 2015 Aug;87(2 Suppl):1323-48.
Initial gene targeting utilized homologous recombination to
insert DNA into a specific locus.
This process is extremely inefficient.
Reprod Biol Endocrinol. 2014 Nov 24;12:108.
The process can be made more efficient by
cutting the DNA and stimulating homology
directed repair

5. Zinc Finger Nucleases: Targeting the Genome
The first specific mechanism to drive a nuclease to a
specific region of the genome.
Made by fusing the Fok I endonuclease domain with
DNA binding zinc fingers.
Zinc fingers are designed in modules of 2, each
recognizing 6 bps. These modules can be strung
together to generate a zinc finger protein that will
recognize as specific sequence in the DNA ( ~24 bp).
Fok 1 is a non-specific endonuclease that has to
dimerize in order to cleave the DNA.
You need to 2 Zinc Finger/Fok1 molecules, each
targeting opposite strands to create a very specific
cleavage site in the DNA.

6. Zinc Finger Nucleases increase the rate of
recombination
Nature 435, 646–651

7. Zinc Finger Nucleases are not very practical
Designing Zinc Finger nucleases with proper specificity is challenging and requires expertise.
Furthermore, zinc finger nucleases have been patented limiting the competition of commercial providers.
Cost of getting a set of zinc finger nucleases is $3000-$7000.

8. TALENs
• Transcription activator-like effector nucleases
J Clin Invest. 2014;124(10):4154-4161
Uses the same conceptual approach as zinc
finger nucleases but replaced the DNA
binding activity of zinc fingers with tale
proteins

9. TALE proteins have a code of specificity
Nature Biotechnology 29, 143–148 (2011)
TALE proteins are bacterial transcriptional activators used to
regulate genes in plants the bacteria infect.
These TALE proteins have a repeat domain of 33-35 bp. Each
repeat recognizes a single base.
A dinucleotide sequence in the middle of the repeat dictates
the recognized base.

10. TALENs increased the practical usage of gene
targeting
Not patented.
Labs generated kits of the different targeting
repeats that could be easily assembled.
No specialized expertise needed for design.
Could be assembled in any lab at relatively low
cost.
Still need to make two unique protein
encoding plasmids for each site you want to
target.

11. CRISPR revolution- hijacking the bacterial
immune system
Foreign DNA will get inserted into a specific locus of the bacterial
genome called the Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR). This locus represents a series of short
repeats broken up by spacers. The spacers are actually foreign
pieces of DNA that have previously integrated into the locus.
The CRISPR locus produces a transcript which gets processed to
create small RNA molecules that contain the spacer and a repeat.
This RNA is called the crRNA.
For type II CRISPR systems, the crRNA interacts with a second RNA
called the tracrRNA and the Cas9 protein.
Together this complex uses the spacer RNA sequence to guide the
complex to foreign DNA. The Cas9 protein then cleaves the DNA
Biochimie.Volume 117, October 2015, Pages 119-128

12. CRISPR targets a DNA sequence
Through RNA/DNA base pairing- the spacer element
interacts with DNA with complimentary sequence
forming an R-loop. An R-loop is three standed
nucleic acid with an RNA-DNA compliment and a
single stranded DNA.
The tracrRNA is needed to form a complex with Cas9
Cas9 is an endonuclease which has two separate
endonuclease domains (HNH & RuvC) which cleave
each strand of the DNA separately.
The carboxy terminus of Cas9 must interact with a
specific DNA element to become activated called the
PAM site.
The PAM site consists of an NGG.
Volume 23, Issue 4, p225–232, April 2015

13. Utilizing this system to target DNA cleavage
There are 3 components to bacterial Cas9/CRISPR system:
1) crRNA- the only part that changes between target sites
2) tracrRNA
3) Cas9
The Spacer sequence is the only part that needs to be
adjusted to target Cas9 cleavage

14. Simplifying the systems into two modules
Combining the crRNA and tracrRNA into one RNA called the
single guide RNA (sgRNA).
Simply turned the junction point into a hairpin.
It can now be encoded by one gene
Molecular Cell. Volume 56, Issue 2, p333–339, 23 October 2014

15. The CRISPR/Cas9 system makes it incredibly
easy to target a nuclease to DNA
Several systems developed in which small
oligos can be easily cloned into a plasmid to
make a guide RNA.
Some of these systems already have Cas9
expression cassettes making it a 1 plasmid
system.
Easy 1 step cloning.
Inexpensive and quick
Target anywhere in the genome with an NGG
site.
All things being random, should be present
every 16 bp
In humans- averages to every 42 bp.
https://www.biocat.com/genomics/genome-engineering/crispr-cas9-smartnuclease-genome-engineering-system-
vector-based

16. The spacer region of sgRNA does not need
100 % base pairing
Nature Methods 12, 1150–1156 (2015)
Similar to miRNA/siRNA, there is a seed sequence that is
more critical in driving the specificity of the complex to
targets

17. Different Cas proteins can modify gene
targeting
Increases potential cut sites (ex: Cas9 needs
GG, Cas12a needs YTT)
Potentially better recombination efficiency
with Cas12a as the cut site is not in the
seed region
Can target RNA.

18. CRISPR has off target
effects
Nature Biotechnology 33, 187–197 (2015)
CRISPR recruits Cas9 to potentially hundreds of sites
across the genome
Read counts suggesting how often
these off target sites are hit

19. Using a nickase to drive HDR with less off
target effects
Cell. Volume 154, Issue 6, p1380–1389, 12 September 2013
Mutations (D10A) to HNH function of Cas9 cause single strand nicks in the
DNA.
Using two sgRNAs to drive Cas9D10A to two close sites can cause a double
strand break.

20. CRISPR initiates indels at target sites
https://www.addgene.org/crispr/guide/
Cas9 mediated DNA cleavage is often repaired by NHEJ.
Using the CRISPR/Cas9 systems, this almost always results an
insertion or deletion of base pairs at the cut site.
When targeted to coding regions, often results in frameshifts that can
cause knockout of the targeted gene if done in the proper region of
the cds.

21. NHEJ competes with HDR to limit the
efficiency of gene editing
Biology Open 2014 3: 271-280;
The inefficiency of HDR in gene editing serves as a major
road block for its use in therapy and animal generation

22. Improving CRISPR through fusion proteins
Fusion of Cas9 to hRad51 mutants improves homology directed repair.
hRad51 directs HDR to single strand DNA (improves HDR at nicked sites).
Mutants can limit “perfect repair”

23. CRISPR Prime to improve editing
CRISPR Prime uses the Cas9 nickase fused to a reverse transcriptase
domain along with a special guide RNA called a pegRNA. The pegRNA is
a guide domain along with a template to modify the cut site.
The 5’ Flap gets chewed back by enzymes associated with DNA
replication (okazaki fragments).
Improved efficiency with lower off target effects. Efficiencies as high as
40%.
Nature. 2019 Oct 21.
Use an additional gRNA to nick non-edited
strand.

24. Using CRISPR to do more than edit the
genome
dCas9 is a version of the Cas9 that has no
endonuclease activity
dCas9 can serve has a protein cargo ship for fusion
partners to modify epigenome or act as a steric
hindrance to DNA binding.
Integr Biol (Camb). 2017 Feb 20;9(2):109-122.

25. dCas9 to regulate transcription
Fusions of dCas9 to KRAB transcriptional repressors
or VP16 transcriptional activators allows for precise
targeting to almost anywhere in the genome.

26. dCas9 can be used to edit the epigenome
Fusions of dCas9 to either
tet or DNMT enzymes
allows for epigenomic
editing
Cell. Volume 167, Issue 1, p233–247.e17, 22 September 2016

27. dCas9 DNMT3 can target methylation
Cell. Volume 167, Issue 1, p233–247.e17, 22 September 2016
The locus only the presence of Dox (inducible system) and
the proper sgRNA is able to drive methylation.

28. Base Editing- CRISPR fusion to base modifying
enzymes
C→T, G→A, A→G, and T→C
Four type of base editing tools
Uses deamination to convert C-T using
APOBEC protein.
Or
Uses TadA to convert A to I(G)
Science 27 Oct 2017:
Vol. 358, Issue 6362, pp. 432-433

29. Directing base editing using
different fusion proteins
Different enzyme variants have different target preferences within the R-
loop.
Use of the PAM site and the enzyme directs which base pairs are most likely
to be edited

30. Summary of Gene Editing techniques