CRISPR/Cas9 TALEN ZFN RNAi

2. OUTLINE OF PRESENTATION
♦ Genome Engineering (from old to new)
♦ CRISPR/Cas9 Technology
♦ Applications
♦ Current Limitations
♦ Article

3. GENOME ENGINEERING
♦ What is the meaning of ‘’Genome Engineering’’ ?
- The process to transfer a pieces of DNA from
one organism to another. (Transgenic organisms!!)
Figure 1: A simple illustration of genetic material transfer
between species.

4. Example
♦ Fireflies have ‘luciferase’ genes that related to
emit light at dark.
♦ Scientists transfered this gene to tobacco plant to
give emiting light ability to tobacco plant in 1986.
Figure 2: Luciferase gene is transferred to tobacco plant. Then,
tobacco started to shine at dark.

5. But, How?
♦ Vectors was used to transfer a gene to a
different genome(host genome).
- Vectors are the tools that help to carry
one piece of DNA to host genome.
- A vector may be bacterial plasmid or
viruses.
Figure 3: extra-chromasomal part of
bacteria = plasmid

6. Figure 4: Basic steps of gene insertion into plasmid. The process based on isolation of desired
sequence of DNA and making multiple copies of it in vivo is called as molecular cloning.

7. Steps in Simply;
1) DNA carrying the
interested gene is taken
form a cell.
2) Gene is inserted into host
cell inside of plasmid.
3) Thr host cell containing
recombinant DNA is
multiplied.(molecular
cloning)
4) Then, your desired protein
sequenced in inserted
gene piece is produced by
recombinant host cell.
Figure 5: preparation of recombinant cell.

8. Figure 6: The picture is demostrated the insertion of related gene into plasmid.
Three different way can be used to put a gene into plasmid. They are shotgun
cloning, PCR products cloning and cDNA cloning seen in the figure.

9. ♦ The methods are based on removing desired
gene pieces from DNA and putting the piece into a
vehicle to expression.
♦ But, how can we cut the genome in specific
part?
- Uses Artificial Nucleases
* ZFN(Zinc Finger Nucleases)
* TALEN(Transcription Activator Like EndoNuclease)
- Or, CRISPR-Cas9

10. ZFN
♦ The first genome-editing tools
that uses customizable
endonucleases.
♦ ZFNs are artificial and made up of
a chain of proteins called zinc fingers
with an enzyme Fok1 attached to
the end of this chain
♦ Zinc Fingers recognizes specific
triplets of DNA bases and Fork1-a
kind of bacterial endonuclease- cuts
the DNA at recognized sequence.
♦Double-stranded breaks(DSBs) are
occured by Fork1 restriction activity.
Figure 7: Restriction mechanism of Zinc
Finger Nuclease

11. ♦ DSBs are important for site-specific mutagenesis
and they stimulate DNA-repairing processes in cell.
- Homologous Recmbination
- Non-Homologous End Joining(NHEJ)
Figure 8: Manifulations with ZFN is seen on figure. In this process, if there is no donor
DNA the repairing system tend to make NHEJ. If there is a donor, it can be used as
template to complete genome(Homologous directed Repair)

12. Figure 9: ZFN-mediated genome editing takes place in the nucleus when a ZFN pair targeting
the user’s gene of interest is delivered into a parental cell line, either by transfection,
electroporation or viral delivery

13. TALEN
♦ They are made by fusing a TAL effector DNA-
binding domain to a DNA cleavage domain (a
nuclease which cuts DNA strands).
♦ Like ZFN, TALEN is used DNA-binding motifs to
cleave DNA in specific sites, but each domain
recognizes single nucleotide (triplets are recognized
in ZFN ).
♦ Transfection efficiency changes due to organism.
To increase efficiency, different strategies may be
used such as electroporation, sending inside
plasmid, sending in mRNA transcript form…

14. Figure 10: TALEN. Like ZFN, Fok1 is used in TALEN,as well. But, in there, DNA binding
domains recognizes single nucleotides instead of triplets.

15. (+) Pros;
♦ Rapid disturption, integration of an loci.
♦ It can apply to all species
♦ Fast animal genome engineering
- the fastest method to obtain knock out
animal(about in 2-3 month)
♦ high mutation rate (about 15% in animals)
♦ No antibiotic selection was required to screening
(if DSB is not repaired, the cells won’t be survival)

16. (-) Cons;
♦ sensitive to DNA modifications(methylation)
♦ Off-target effects created by endonucleases
can be toxic to cell and the prediction or
monitoring of side-effect is difficult.
♦ Availability of desired repairing system in cell
line is a big limiting factor.
♦ To overcome this, endonucleases are fused
with enzymatic domains such as side-specific
recombinase or transposase to catalase the DNA
integration, inversion or excision.

17. CRISPR-Cas9
♦ It is the part of immune system of certain
bacteria against viruses.
♦ When viruses(bacteriophase) insert into bacteria,
it integrates its genome into bacterial genome to
express own viral proteins.
♦ In this situation, if the bacteria survives, the
genome of viruse remains bacterial genome.
♦ After that, if bacteria exposes the same virus,
cas9 proteins recognize the virus DNA, then disrupt
it to prevent repruduction of virus.
♦ CRISPR technology is based on this strategy.

18. ♦ Unlike ZFN/TALEN, Cas9 uses complementary
RNA to achieve sequence specifity.
♦ Cas9 is a nuclease that is no need to fuse
addition of any protein.
♦ Cas9 can be programmed to target nearly any
gene in the genome by synthesizing a guide RNA
(gRNA) molecule, which is complementary to
the target sequence.
♦ Recently, an alternative CRISPR proteins is
founded which is called as Cpf-1.

19. Figure 11: CISPR-cas9 protein is seen on figure. It makes DSB due to guide RNA(gRNA)
sequence. PAM(protospacer adjacent motif) is required to facilitate recognition of target
genome. When gRNA sequence and genome sequence is matched, cas9 cleavages the
genome.

20. Cpf1 vs Cas9
♦ Both two proteins funtion in the same principle.
♦ Like Cas9, Cpf1 is a nuclease paired with a gRNA
composed of a variable targeting region and a
constant structural region.
♦ But; gRNA for CRISPR-cpf1 is shorter than gRNA
for CRISPR-cas9.(about 45 nucleotides for Cpf1 vs.
about 110 nucleotides for Cas9)
♦ This length may make in vitro synthesis of gRNA
cheaper and easier.

21. ♦ Cpf1 uses a different PAM than Cas9. It recognizes a T-
rich PAM, TTTN, but on the 5' side of the guide. This
makes it distinct from Cas9, which uses an NGG PAM on
the 3' side.
♦ So, it makes cpf1 a useful tool at AT-rich genomes
because cas9 needs at least one G to recognition.
♦ While Cas9 generates a blunt-end cut, Cpf1 generates
an overhanging cut.
♦ There is some evidence that this overhanging cut may
facilitate non-homologous end joining-mediated knock-in
of target genes; however, more research into this idea is
necessary.

22. Figure 12: Cpf1 and Cas9 proteins are seen on schema. Both contains gRNA but it is shorter in cpf1
protein. The PAM sequences are different in two proteins. It recognizes a T-rich PAM, TTTN, but on the
5' side of the guide. This makes it distinct from Cas9, which uses an NGG PAM on the 3' side. The last
difference between them is the double stranded break styles. Cas9 makes blunt end breaks unlike
cpf1(overhanging breaks).

23. Delivery of Cas9 into nucleus
♦ To achieve CRISPR-mediated gene editing, there
must ultimately be a functional Cas9–gRNA
ribonucleoprotein (RNP) complex present in the cell
nucleus.
♦ But, delivering a large protein across the cell
membrane can be extremely challenging.
♦ Thus, in addition to this protein delivery approach,
research has focused on delivering genetic material
to instruct the target cell to produce its own Cas9
protein in situ.

24. ♦ Delivering either DNA (in the form of either a
plasmid or encoded in a viral genome) or mRNA
can lead to expression of the Cas9 protein inside
of a target cell, and ultimately result in Cas9-
mediated gene editing.

25. DNA mRNA PROTEIN
insert cas9-encoding gene
into plasmid
Cas9 is expressed by
ribosomes found in the
cytoplasm.
The most difficult form of
delivery because of big
structure of protein.
Problems in passing
through cell membrane,
nucleus membrane.
Faster than plasmid
delivery because of quicker
onset
In vitro produced cas9 and
gRNA are delivered to cell
then wait for occuring RNP
complex.
Plasmid promotor may be
incompatible with
mammalian cells.
Transient protein
expression should be
controlled tightly
Problem in obtaining
protein in pure form.
Expression time is higher
than other models.
mRNA is less stable than
DNA.
If there is problem in
purity, toxic effect may
seen because of endotoxins
it may pros in procedures
that required sustaining
expression but may cause
off-target effects
Requires modifications to
increase mRNA stability
Bacterial protein may cause
side effect in mammalial
immune.
May cause immunogenic
response
Required high cost
Table 1: Cas9 delivery ways.

26. ♦ Cas9 may be delivered as a DNA or mRNA molecule
encoding for the cas9 gene, or it may be delivered as a
functional ribonucleoprotein(RNP).
♦ Regardless of cargo format, the largest challenge lies in
delivering the cargo across the cell membrane.
♦ A variety of viral and non-viral methods have been derived
to achieve successful delivery across the cell membrane.
- AAV (adenoassociated virus)
- AuNP (gold nanoparticle)
- Cas9 (CRISPR-associated protein 9)
- CPP(cell-penetrating peptide)
- LNP( lipid nanoparticle)
- NLS (nuclear localization sequence)

27. Figure 13: The vehicles of cas9 delivery.

28. Viral Delivery
♦ remove the rep genes in virus that cause
replication of viral genome in host.
♦ replace rep gene with desired gene.
♦ the virus is still able to enter a host cell and
induce the expression of the transgene, but is no
longer able to replicate itself or spread to new cells
♦ The most common used viruses are adeno-
associated virus(AAV)

29. ♦ there is a risk that the transgene integration into
the target genome could inadvertently disrupt the
expression of a vital gene.
♦ The maximum capacity of an AAV is around 4.7
kb.(gene limit)
♦ Thus, designing a virus to encode such a large gene
as cas9 along with its promoter and gRNA is
challenging.
♦ One solution to this problem is to pack gRNA and
Cas9 in separate viral vectors.

30. Physical methods
♦ based on the disruption of physical barriers to
send delivery without causing permanent
damage.
♦ Electroporation is the technique that exerts
strong electric field to cell membrane for
increasing the permeability.
♦ This method is proper for in vitro studies.

31. ♦ Microinjection uses micron-scale needles to
simply pierce the cell membrane and directly inject
the cargo.
♦ Microinjection offers high efficiency, precisely
controlled dosage, and a guarantee that the cargo is
delivered exactly to the intended site.
♦ Each individual cell of the target tissue must be
injected manually and it is highly impractical to
perform in a high-throughput manner

32. Chemical Delivery
♦ based on modifying the target to pass through cell
barriers.
♦ Cell penetrating peptides (CPP) are short peptide
sequences that can cross the cell membrane through
either direct penetration, translocation mediated by
endocytosis, or formation of micelles.
♦ Although the exact mechanisms are not fully
understood, it is believed that the composition of
CPPs of either highly abundant positively charged
amino acids or alternating polar and nonpolar amino
acids is responsible for their activity

33. ♦ Nuclear localization sequences (NLSs) are
naturally occurring sequences that tag proteins
that are synthesized in the cytoplasm for
transport into the nucleus.
♦ NLSs are made up of polyarginine or poly-
lysine chains conjugated to the protein surface
and stimulate nuclear import signals to allow
passage through the nuclear membrane

34. APPLICATIONS

35. Vaccination of bacterial strains
♦ In bacteria-based industries, including dairy
production and bacteria-based biorefineries,
contamination of the fermenter with bacteriophages
causes a catastrophe.
♦ Traditionally, attempts to protect industrial bacteria
from bacteriophage attacks focused on modification of
bacterial innate immune systems.
♦ The strategies include interference of bacteriophage
adsorption, prevention of phage DNA or RNA injection,
digestion of infecting nucleic acid by R-M systems, and
enhancement of abortive infection.

36. ♦ Vaccination of bacteria against their viruses was the
first application utilizing full CRISPR/Cas systems. This
application borrows unmodified, native function of the
CRISPR/Cas systems from the host bacteria.
♦ First proved the role of CRISPR/Cas systems in
prokaryotic acquired immunity and demonstrated
enhancement of resistance against bacteriophages in a
yogurt and cheese starter bacteria S. thermophilus.
♦Artificial introduction of spacers targeting
bacteriophage DNAs into its CRISPR array gave
bacteriophage-resistance to the engineered S.
thermophilus

37. Extracting HIV-1
♦ Nowadays, HIV-1 hosts
must use an anti-retroviral
drug coctail to maintain
their life.
♦ A research group used
CRISPR/cas9 technology to
remove HIV-1 containing
gene from a living mouse
genome.
♦ They have cleaned the
whole genome of mouse
just in one therapy.
Figure 14: CRISPR for removing HIV.

38. Personal Application
♦ Embryonic stem cells are edited with the
CRISPR technique, and then, re-injected into the
patient.
♦ In this method, each person is treated
according to their genetic characteristics, and
their faulty genes will be modified directly.

39. Cancer
♦ Cancer is generated by oncogenes or tumour
suppressor genes for various genetic and epigenetic
reasons.
♦ Therefore, the CRISPR system could potentially treat
the disease by generating accurate mutations through
turning off the oncogenes or turning on the tumour
suppressor genes.
♦ Given that the telomeres become activated in various
cancers and cause cancer cells immortality, turning off
telomerase by CRISPR has also been proposed as another
potential method for cancer treatment

40. GMO
♦ Modification of genome is simple when
CRISPR is used.
♦ It is important in producing resistant plants
against pesticides, bugs, drough, cold, hot…
♦ The other usage is to producing disease-
resistant cattles.

41. Design Your Dream Baby
♦ the scientists may adjust your baby how you
are want.
♦ Tall/short in height; blonde/ginger hair,
green/blue eyes… what you want.

42. LIMITATIONS
♦ Difficulty to deliver cas9 and gRNA to the
target in living organism.
♦ Hard to design a proper gRNA. Because sgRNA
unique target sequences should be different
from any other sites in the genome by at least
three nucleotides in 20-nucleotide sequences.
♦ otherwise, it may cause off-target effect.

43. ♦ In living organism genome, the treated DNA may
be reverted by natural mechanisms.
♦ Cas9 protein is a bacterial protein that may be
cause immunotoxic effects on eukaryotes especially
mammalians.
♦ Recent researches shows that there are some
viruses that are resistant to Cas9 proteins. So,
scientists are try to develop new strategies to
overcome.

44. What about Ethics?
♦ It’s one thing to remove life-impacting
diseases before birth – it’s quite another for
parents to be able to design their babies to be
stronger, faster or better looking.
♦ Even if you accept that this is something
people should be allowed to do, the chances are
this would be heavily commercialised, ensuring
only the rich could afford all the extra life
advantages this would afford, massively
affecting inequality.

45. YOUR THOUGHTS?

46. LET’S LOOK AT SOME ARTICLES

48. RNA interference (RNAi)
♦ a regulatory mechanism of most eukaryotic
cells
♦ commonly achieved using short-interfering
RNAs (siRNAs) or short-hairpin RNAs (shRNAs)
♦ the introduced RNA is loaded into the RNA
induced silencing complex (RISC), which in turn
promotes the degradation of perfectly
complementary target mRNA

49. ♦ Using this approach, target mRNA and
subsequently target protein levels can be
reduced postranscriptionally
♦!! So, it is a good approach to ‘gene silencing’
in protein level.
♦ No degradation of genome!!b JUST RNA is
diced.

50. BUT;
♦ the RNAi machinery appears to be mostly
active in the cytoplasm, nuclear transcripts
♦ for example long non-coding
RNAs(lncRNAs)** - can be more difficult to
effectively target
**Long non-coding RNAs (long ncRNAs, lncRNA) are
defined as transcripts longer than 200 nucleotides that
are not translated into protein.**

51. ♦ Moreover, it was recently shown that the
transcription of lncRNA itself can have functional
consequences.
♦ Hence reducing lncRNAs at a post-
transcriptional level via RNAi may not always
reveal their full loss-of-function phenotype

52. ADVANTAGES;
♦ the silencing machinery is present in
practically every mammalian somatic cell.
♦ So, no prior genetic manipulation of the
target cell line is required, and a simple siRNA
transfection can result in a loss-of-function
phenotype.

53. Specificity & Off-target Effects of RNAi
♦ A big problem!!
♦ siRNAs can induce silencing of non-target mRNAs
with limited sequence complementarity, often via
interactions with the 3′UTR
♦ These interactions occur when short regions of
the mRNA 3′-UTR contain imperfect matches to the
small RNA, triggering translational repression
and/or degradation

54. ♦ depending on the sequence, one siRNA can
potentially repress hundreds of transcripts
♦ These off-target effects appear to be dosage
dependent and phenotypes derived from them can
be dominant over the intended on-target
phenotypes
♦ Moreover, the artificial introduction of siRNAs or
shRNAs into a target cell line can cause non-
sequence specific off-target effects

55. ♦ RNAi uses a natural pathway that regulates
cellular gene expression levels, where the
system is governed by endogenous microRNAs.
♦ Flooding the system with exogenous
sequences can displace microRNAs from RISC.
♦ This can impair the functions of endogenous
microRNAs, leading to alterations in gene
transcript levels and consequently to off-target
phenotypes

56. TALE Transcriptional Repressor
♦ Gene silencing by means of transcriptional
repression can be achieved using KRAB-TALEs,
♦ the transcriptional repressor Krüppel
associated box (KRAB) domain is fused to DBDs
that target the transcription start site (TSS)

57. ♦ A crucial difference of this approach when
compared to RNAi is that TALE repression
prevents the transcription of targeted genes in
the nucleus, while RNAi degrades them post-
transcriptionally in the cytoplasm
♦ TALE-repression mimics the hypomorphic
effect of RNAi, such that gene function is
reduced but not shut off permanently, as it is
the case for TALENs.

58. Specificity and off-target effects of
TALEN technologies
♦ Because of the requirement of FokI to
dimerize in order to have nuclease activity, no
single TALEN can ever introduce a double strand
break into a target site.
♦ two TALENs have to off-target to adjacent
sites in order to introduce an unspecific double
strand break into the DNA.

59. ♦ Consequently, the off-target activity observed
from TALENs is typically low
♦ Unlike TALENs, TALE transcriptional repressors
function as monomers and can hence mediate
off-target effects autonomously.

60. CRISPR interference (CRIPSRi)
♦ Similar to TALE transcriptional repressors
♦ an enzymatically inactive version of Cas9
(deadCas9 or dCas9) is fused to a repressor
domain (e.g. KRAB), triggering heterochromatin
mediated gene silencing at the TSS
♦ leads to transcriptional reduction of the target
RNA, similar to TALE transcriptional repression.

61. ♦ In comparison to RNAi, CRISPRi appears to
produce a more consistent and robust
knockdown given the same number of effector
RNAs.

62. Disadvantages;
♦ not ensure that a given sgRNA may engage and be
active on a given target site.
♦ the accessibility and location of the TSS is critical don’t
be known:
♦ Cas9 needs to physically access its target site. This
process has been shown to be dependent on chromatin
accessibility
♦ Hence genomic regions with closed chromatin states
may prevent binding and thus the function of Cas9.

63. CRISPR activation (CRISPRa)
♦ Gain-of-function studies in mammalian cells
have traditionally been carried out by
overexpressing transgenic open reading frames
(ORFs or cDNAs) in target cells.
♦ based on cloning an ORF behind a promoter
and introducing it into target cells.

64. Choosing the right tool for the job:
KNOCKOUT
♦ For many applications, a full knockout may be
desirable, and perhaps even required.
♦ For instance, in cases of slow protein
degradation, reduced transcript levels - as
caused by knockdown approaches - may not
necessarily result in low protein levels.

65. ♦ Moreover, if a gene product is not rate
limiting, even strongly reduced protein levels
may not produce a loss-of-function phenotype.
♦ A knockout on the other hand would sooner
or later lead to full depletion of functional
protein.

66. ♦ A significant advantage of Cas9 nuclease mediated
genome editing compared to transcriptional repression
via CRISPRi, is that it targets exonic regions rather than
TSSs.
♦Why is this important?
-First of all, exons are generally very well annotated
and can be deduced from transcriptome sequencing data.
-More importantly though, genome editing can
disrupt exons that are shared by all variants of a given
gene, even if these are transcribed from separate TSSs.
- Like this, all transcript variants and even whole
gene families can be knocked out using a single sgRNA
against exons that are conserved between all family
members.

67. ♦ Most common laboratory cancer cell lines exhibit
changes in ploidy, with many lines carrying four or
more copies of a chromosome
♦ In these cases, true knockout(fully distruption of
target gene function) phenotypes may only emerge
once every functional copy of the target gene is
mutated.
♦ In practice, this may be difficult - although not
impossible - to accomplish.
♦ Depending on the experiment, it may be
imperative to sequence the target loci to verify
homozygous knockout when generating mutant
clonal cell lines.

68. Gene knockdown
♦ While not always capable of disrupting gene
function completely, hypomorphic knockdowns can
offer a number of advantages over knockouts.
♦ Since gene knockdown via RNAi or CRISPRi does
not depend on frame shift mutations or ploidy,
issues related to genetic heterogeneity are
minimized.
♦ Moreover, gene knockdowns are reversible, a
feature that has proven very useful in the past

69. ♦Furthermore, partial gene knockdown allows
researchers to study the function of essential genes
whose knockout would otherwise be cell-lethal.
♦ Additionally, applications such as drug target
discovery could benefit from phenotypic
hypomorphs, since an RNAi or CRISPRi knockdown -
rather than a knockout - may mimic the effects of
an inhibitive drug more closely.
♦ However, in some cases, a full knockout may
represent an idealized loss-of-function phenotype
for the most potent inhibitive drugs.

71. - Bacterial populations in the biosphere are
confronted by 1030
phage infections each day.
- Bacteria have to develop some mechanism to
survive:
- Restriction modifications
- Endonucleases. Protect own DNA by methyl addition.
- Superinfection exclusion
- Preexisting viral infection prevents other infection by some
proteins(mechanism is not known)
- Abortive infection mechanisms.
- Suicide of infected cell to stop spreading of virus to protect
remaining population ( very honorable ☺)
- CRISPR/Cas9 is a kind of immune system in
bacterial against foreign nucleic acids.

72. - CRISPR locus serves as genetic library of previous
viral infectious that cell survives.
- But…..
- Phages found ways to escape from Cas9 system
in bacteria
- One of the way is that phages create point
mutations in their genomes.
- Changing in one base cause reduction in binding
affinity of guide-RNA complex
- So, phaga escapes from CRISPR-mediated
destruction.

73. - However;
- bacteria rapidly acquire new spacers in
response to these escape mutants through a
process known as primed adaptation
- This ability of bacterial populations to quickly
retarget phage escape mutants with new
spacers acquired by different cells allows them
to drive phages to extinction despite their
mutational evasion.

74. Anti-CRISPR
- In contrast to mutational drift, anti-CRISPR
proteins provide phages with an active counter-
defense system that inactivates CRISPR-Cas
interference complexes in a sequence
independent manner.
- The first anti-CRISPR proteins, described in 2013,
were found in a group of closely related phages
that infect Pseudomonas aeruginosa
- The phage was containing a unique locus that
inactivates CRISPR complex.

75. - - This locus encoded ten different gene
families that were all small (150–450
nucleotides) and of unknown function.
- Each gene was tested for the ability to inhibit
type CRISPR-Cas activity in P. aeruginosa, and
five distinct genes (acrF1–acrF5) were shown
to have this activity

77. -The nine anti-CRISPR protein families initially
identified were restricted to the Pseudomonas
genus and did not share any common sequence
features.
-Thus, it was difficult to predict whether anti-
CRISPRs were widespread among bacteria or a
unique feature of type I systems in Pseudomonas

78. Mechanism
- There are numerous steps of CRISPR-Cas activity
that could be subject to inhibition by anti-CRISPR
proteins.
- For example, anti- CRISPRs could prevent the
acquisition of
- new CRISPR spacers,
- block expression of Cas proteins,
- inhibit guide-RNA transcription or processing,
- prevent the assembly of the active CRISPR-Cas
complex,
- inhibit binding to the foreign DNA element, or block
cleavage activity.

79. - There are 22 unique families of anti-CRISPR
proteins which is identified
- Each of these protein families encoding small
proteins that are unrelated to any proteins of
known function.
- This has prevented the prediction of protein
mechanisms in the absence of experimental
evidence.

80. Current findings;
-The six anti-CRISPRs for which activities have
been described can be roughly grouped into two
mechanistic types:
♦disrupt DNA binding and those that inhibit
target sequence cleavage.
♦ No anti-CRISPRs that affect the expression
of Cas genes or expression and processing of
guide-RNAs have been discovered to date.

81. - The most common mechanism identified so
far is interference with DNA binding activity
through a direct interaction with the CRISPR-
Cas complex.
- The precise mechanisms by which anti-
CRISPRs block DNA binding and allow it to
escape recognition vary among the anti-
CRISPR proteins

82. Future Directions about anti-CRISPR
- In genome editing applications, anti-CRISPRs may
provide a valuable ‘‘off switch’’ for Cas9 activity
for therapeutic uses and gene drives.
- One concern of CRISPR-Cas gene editing
technology is the limited ability to control its
activity after it has been delivered to the cell.
- Cas9 is generally active as long as it is present in
the cell and has access to its guide RNA molecule,
which can lead to off-target mutations.

83. - Anti-CRISPRs can potentially be exploited to
target Cas9 activity to particular tissues or
organs, to particular points of the cell cycle, or
to limit the amount of time it is active.
- The efficacy of anti-CRISPRs in these types of
applications and in the control of Cas9 activity
in whole organisms has yet to be explored.

84. -Anti-CRISPRs may also prove to be a useful
addition to phage therapy protocols for the
treatment of bacterial infections.
- Because many human pathogens encode
CRISPR-Cas systems, phages used for gene
therapy could be outfitted with a variety of anti-
CRISPRs to expand their host range and prevent
the bacterial adaptive immune response.

86. REFERENCES
Gaj, T., Gersbach, C. A., & Barbas, C. F. (2013). ZFN, TALEN and CRISPR/Cas-based methods for genome
engineering. Trends in Biotechnology, 31(7), 397–405.
Wang, H.X. et al. (2017) CRISPR/Cas9-based genome editing for disease modeling and therapy:
challenges and opportunities for nonviral delivery. Chem. Rev. 117, 9874–9906
Ramakrishna, S. et al. (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9
protein and guide RNA. Genome Res. 24, 1020–1027
Jiang, C. et al. (2017) A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA
and pcsk9 in vivo. Cell Res. 27, 440–443
Jinek, M. et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial
immunity. Science 337,816–821
Urnov, F.D. et al. (2005) Highly efficient endogenous human gene correction using designed zinc-finger
nucleases. Nature 435, 646–651
Cong, L. et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823

87. L.A. Marraffini, E.J. Sontheimer CRISPR interference limits horizontal gene transfer in
staphylococci by targeting DNA Science, 322 (5909) (2008), pp. 1843-1845
L.S. Qi, M.H. Larson, L.A. Gilbert, J.A. Doudna, J.S. Weissman, A.P. Arkin, et al.
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene
expression Cell, 152 (5) (2013), pp. 1173-1183
D. Bikard, W. Jiang, P. Samai, A. Hochschild, F. Zhang, L.A. Marraffini
Programmable repression and activation of bacterial gene expression using an
engineered CRISPR-Cas system Nucleic Acids Res, 41 (15) (2013), pp. 7429-7437
S. Kiani, A. Chavez, M. Tuttle, R.N. Hall, R. Chari, D. Ter-Ovanesyan, et al. Cas9 gRNA
engineering for genome editing, activation and repression Nat Methods, 12 (11)
(2015), pp. 1051-1054
W. Jiang, D. Bikard, D. Cox, F. Zhang, L.A. Marraffini RNA-guided editing of bacterial
genomes using CRISPR-Cas systems Nat Biotechnol, 31 (3) (2013), pp. 233-239
L. Cong, F.A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, et al. Multiplex genome
engineering using CRISPR/Cas systems Science, 339 (6121) (2013), pp. 819-823
D. Bikard, W. Jiang, P. Samai, A. Hochschild, F.
Zhang, L.A. Marraffini
Programmable repression and activation of
bacterial gene expression using an engineered
CRISPR-Cas system
Nucleic Acids Res, 41 (15) (2013), pp. 7429-
7437