1. Genome editing

2. Genome editing is a technique where DNA is inserted, replaced or removed from a genome using artificially engineered nucleases

3. Why Genome editing? Accelerate basic research Target gene mutation
Knockout gene
Study gene function
Disease model
Gene therapy
Replace defective genes
Fix specific cell types

4. Genome editing
Most popular genome engineering techniques apply DNA-cutting enzymes/complexes that generate targeted double-strand cuts
They are repaired by the host cells by either the error-prone, non-homologous end joining repair system (NHEJ), or the homologous recombination-based double-strand break repair pathway (HDR).
The most frequent application of these endonuclease-based tools is the study of gene function through the inactivation of the target gene
In addition, by providing a repair template, these systems allow for gene replacement strategies by taking advantage of the host cell's dsDNA break homologous repair system.
These new methods have tremendous potential towards the development of more accurate cellular and humanized laboratory animal models for various pathological conditions
Moreover, these endonuclease-based genetic engineering techniques are being developed as therapeutic agents to cure human monogenic diseases

5. Genome editing
In general, genome editing tools using DSB nuclease-driven reactions can be divided into two groups.
The first group consists of MNs, ZFNs and TALENs, which achieve sequence-specific DNA-binding via protein-DNA interactions.
The second group is comprised of two sub-groups: (i) CRISPR/Cas9 and targetrons, which are RNA-guided systems and (ii) DNA-based-guided systems.

6. Genome editing Genome editing tools include meganucleases (MNs)
zinc finger nucleases (ZFNs)
transcription activator-like effector nucleases (TALENs)
clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease Cas9
targetrons
All of them can achieve precise genetic modifications by inducing targeted DNA double-strand breaks (DSBs).
Depending on the cell cycle stage, as well as the presence or absence of a repair template with homologous terminal regions, the DSB may then be repaired by either NHEJ or HDR

7. Genome editing

8. Genome editing

9. Genome editing

10. Genome editing: Meganucleases
Meganucleases were the first class of sequence specific nucleases, and they continue to be deployed to achieve complex genome modifications.
An advantage of meganucleases is their size. They are among the smallest nucleases–comprising only 165 aminoacids(aa)- making them amenable to most delivery methods, including vectors with limited cargo capacities.
DNA

11. Genome editing: Meganucleases
Relative to other sequence-specific nucleases, however, meganucleases are challenging to re-design for new target specificity. Redesign is hindered by the non-modular nature of the protein.
For example, within the LAGLIDADG family of meganucleases, the aminoacids responsible for binding DNA overlap with those for DNA cleavage;
Therefore, attempting to alter the DNA-binding domain can affect the enzyme’s catalytic activity. As a result, the use of meganucleases has been limited to naturally occurring meganuclease s(e.g.,I-SceI,I-CreI)or to redesigned nucleases made by groups with expertise.
DNA

12. Genome editing: Meganucleases vs ZNFs or TALENs
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research.
These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a non-specific DNA cleavage domain.
ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone non-homologous end joining or homology-directed repair at specific genomic locations.

13. Genome editing : ZNFs
Fokl = nucleases with a domain that cut DNA in an unspecific way
The “modular assembly” approach involves the use of a pre-selected library of zinc-finger modules generated by selection of large combinatorial libraries or by rational design.
Because zinc-finger domains have been developed that recognize nearly all of the 64 possible nucleotide triplets, pre-selected zinc-finger modules can be linked together in tandem to target DNA sequences that contain a series of these DNA triplets.

14. Genome editing : ZNFs
Like the meganucleases, zinc-finger nucleases are relatively small (300aa per monomer), making them amenable to most delivery methods.
DNA targeting by zinc-finger nucleases is achieved by arrays of zincfingers, each of which typically binds to a nucleotide triplet(3to6).
FokI activity requires dimerization; therefore, to site-specifically cleave DNA, two zinc-finger nucleases are designed to bind to DNA in a tail-to-tail orientation.
Whereas redesigning the zinc-finger DNA-binding domain is not as difficult as for meganucleases, there are still challenges in achieving new target specificity, mostly due to the influence of context on zinc-finger function.

15. Genome editing : ZNFs
For example, a zinc-finger that recognizes GGG in one array may not recognize this sequence when positioned next to different zinc-fingers. As a result, modular assembly of zinc-fingers has had limited success. One of the more successful methods for redirecting targeting involves screening libraries of three zinc-finger variants to identify those that best recognize and bind to their intended target sequence.
More recently, modular methods for constructing zinc-finger arrays have been successful that use two-finger units to minimize context effects. Consequently, generating functional zinc-finger nucleases is now achievable by most research labs.

16. Genome editing : TALENs

17. Genome editing : TALENs
The discovery of a simple modular DNA recognition code by transcription activator-like effector (TALE) proteins has led to the explosive expansion of an alternative platform for engineering programmable DNA-binding proteins.
TALEs are naturally occurring proteins from the plant pathogenic bacteria genus Xanthomonas, and contain DNA-binding domains composed of a series of 33–35 amino acid repeat domains that each recognizes a single bp.
TALE specificity is determined by two hypervariable amino acids that are known as the repeat-variable diresidues (RVDs).

18. Genome editing : TALENs
Like zinc-fingers, modular TALE repeats are linked together to recognize contiguous DNA sequences. However in contrast to zinc finger proteins, there was no re-engineering of the linkage between repeats necessary to construct long arrays of TALEs with the ability to theoretically address single sites in the genome.

19. Genome editing : CrispR- Cas9
Distinct from the site-specific nucleases described above, the CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/CRISPR-associated (Cas) system has recently emerged as a potentially facile and efficient alternative to ZFNs and TALENs for inducing targeted genetic alterations

20. Genome editing : CrispR- Cas9

21. Genome editing : CrispR- Cas9
Discovery of CRISPR-Cas9
CRISPR= Clustered Regularly Interspersed Short Palindromic Repeats (Dna Repeats)
Cas= CRISPR associated (protein coding sequences)

22. Genome editing : CrispR- Cas9
In bacteria, the CRISPR system provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage.
In the Type II CRISPR/Cas system, short segments of foreign DNA, termed “spacers” are integrated within the CRISPR genomic loci and transcribed and processed into short CRISPR RNA (crRNAs).
These crRNAs anneal to trans-activating crRNAs (tracrRNAs) and direct sequence-specific cleavage and silencing of pathogenic DNA by Cas proteins.

23. Genome editing : CrispR- Cas9
Work has shown that target recognition by the Cas9 protein requires a “seed” sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA- binding region

24. Genome editing : CrispR- Cas9
How bacteria prevent Dna invasion from viruses?

25. Genome editing : CrispR- Cas9
How bacteria prevent Dna invasion from viruses?

26. Genome editing : CrispR- Cas9
How bacteria prevent Dna invasion from viruses?

27. Genome editing : CrispR- Cas9
How bacteria prevent Dna invasion from viruses?

28. Genome editing : CrispR- Cas9
How bacteria prevent Dna invasion from viruses?

29. Genome editing : CrispR- Cas9
From immune system to an engineered technique

30. 2 component system Genome editing : CrispR- Cas9
From immune system to an engineered technique
2 component system

33. Genome editing : CrispR- Cas9
From immune system to an engineered technique
DNA cleavage is based on RNA/DNA pattern and not anymore on Protein/DNA

34. Genome editing : CrispR- Cas9
From immune system to an engineered technique
DNA cleavage is based on RNA/DNA pattern and not anymore on Protein/DNA
Change require only in the 20’ first nucleotides of the gRNA (former crRNA)

35. Genome editing : CrispR- Cas9
From immune system to an engineered technique
DNA cleavage is based on RNA/DNA pattern and not anymore on Protein/DNA
Change require only in the 20’ first nucleotides of the gRNA (former crRNA)
Possibility of targeting multiple DNA sequences at once

36. Genome editing : CrispR- Cas9
From immune system to an engineered technique
DNA cleavage is based on RNA/DNA pattern and not anymore on Protein/DNA
Change require only in the 20’ first nucleotides of the gRNA (former crRNA)
Possibility of targeting multiple DNA sequences at once
Much more easier to target DNA sequence

37. Genome editing : CrispR- Cas9 Some limitations
Off-target: tolerance of Cas9 to mismatches in the RNA guide sequence.
One crucial concern when applying these genetic editing tools is the potential of cleavage at non-targeted sites.
This event can be lethal or generate undesirable mutations resulting in the requirement of extensive screening in order to identify cells with the desired site-specific modifications.

38. Genome editing : CrispR- Cas9 Some limitations
Off-target: tolerance of Cas9 to mismatches in the RNA guide sequence.
Limited by PAM motif
Depend of mismatchs locations, lengths, compositions
Difficult to predict

39. Genome editing : CrispR- Cas9
Variants of Cas9

40. Genome editing : CrispR- Cas9
Variants of Cas9

41. Repress multiple target genes with reversibility
Genome editing : CrispR- Cas9
Variants of Cas9
Repress multiple target genes with reversibility
Fuse Cas9 with activator/repressor/fluorescent domains

42. Repress multiple target genes with reversibility
Genome editing : CrispR- Cas9
Variants of Cas9
Repress multiple target genes with reversibility
Fuse Cas9 with activator/repressor/fluorescent domains

43. Genome editing : CrispR- Cas9
Variants of Cas9

44. Genome editing : CrispR- Cas9 Variants of Cas9
Cpf1 and is now classified as Cas12a, which is well-suited for experiments targeting AT-rich DNA sequences.
Cpf1 nucleases from different bacterial sources recognize slightly different (but AT-rich) PAM sequences.
While FnCpf1 recognizes PAM sequence 5′‐TTN‐3′, AsCpf1 and LbCpf1 recognize 5′‐TTTV‐3′, where V is A, G, or C nucleotide.
As Cpf1 creates a staggered double-strand cut in the target DNA, rather than the blunt-end cut generated by SpCas9.
Also, Cpf1 is smaller than SpCas9 and does not require a tracer RNA. The guide RNA required by Cpf1 is therefore shorter in length, making it more economical to produce. 

45. Genome editing : CrispR- Cas9
Variants of Cas9

46. Genome editing : repair mechanisms
Mechanisms of which pathways is taken is not fully understood

47. Genome editing : repair mechanisms
Non-homologous end joining (NHEJ) functions in all kinds of cells, from bacteria to man.
It is involved in many different processes, such as DNA repair, telomere maintenance, and the insertion into the genome of HIV-1 and repetitive sequences.
NHEJ of double-stranded breaks (DSBs) in DNA is accomplished by a series of proteins that work together to carry out the synapsis, preparation and ligation of the broken DNA ends.
Deficiencies in any one of these proteins results in hypersensitivity to DNA DSB-inducing agents.
Three steps : DNA end-binding and bridging, terminal end processing, and ligation.

48. Genome editing : repair mechanisms
NHEJ can result in frameshift mutations that usually lead to gene disruption or gene knockout and/or the production of nonfunctional truncated proteins;
one exception being when a frameshift mutation was introduced to correct a defective coding sequence in the dystrophin gene

49. Genome editing : repair mechanisms
Usefulness of DNA repair by NHEJ
An effective form of mutagenesis
Diversity of breakpoints repaired by NHEJ = multiple alleles are generated instantly
Make two breaks for large deletions

50. Genome editing : repair mechanisms
Homologies Directed Repairs - HDR
DSBs are immediately resected to generated free 3′ ends, eliminating the possibility of reannealing.
RPA binds the free ends and is replaced by RAD51, which promotes invasion of duplex DNA donors and canonical HDR.
SSO donors can also support efficient alternative HDR at DSBs, via a pathway that is independent of RAD51.

51. Genome editing : repair mechanisms
Usefulness of DNA repair by HDR
Gene therapy
Gene replacement
Genome engineering

54. video

57. The aim of this work was to generate 4 mouse lineages invalided for the Cfap43, Cfap44, FlagC and FlagF genes, respectively, to validate the candidate genes by assessing the spermatogenesis of the corresponding knock-out mice. Additionally, the generation of a knock in (KI) was also carried out consisting of inserting 27 nucleotides coding for the HA tag at the end of the FlagF gene.

58. Reproductive phenotype was studied for both KO mice models
Reproductive phenotype was studied for both KO mice models. Homozygous KO males exhibited complete infertility when mated with WT females. Sperm concentrations fell within the normal values for mouse but all spermatozoa were immotile. For Cfap43−/−, 100% of sperm had a short flagella whereas Cfap44−/− had a flagella of normal length with some moderate morphological abnormalities but with a complete absence of motility

59. Study of monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combined patient-derived and genetically engineered induced pluripotent stem cells (iPSCs) with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene encoding tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural and functional abnormalities associated with TAZ mutation.

63. Single particle tracking revealed the confined diffusion of telomeres, which is occasionally overlaid with a slow directed motion (Figure 5A and Movie S1). To test if CRISPR labeling could affect telomere dynamics, we compared telomere movement labeled by CRISPR or TRF1, one of the major telomeric binding proteins (Wang et al., 2008). We saw very similar mean-squared displacement (MSD) curves using two methods, demonstrating that CRISPR does not disrupt telomere dynamics (Figure 5B).

64. In addition to special genetic elements such as the telomere, CRISPR imaging also allows us to examine the spatiotemporal dynamics of protein-encoding DNA sequences in live cells. By tagging MUC4 exon 2 and intron 3 simultaneously using two sgRNAs, we measured the position of MUC4 loci by approximating the shape of the nucleus as an oval (Figure 6A). The distribution of normalized MUC4 radial position peaked near the nuclear envelope (Figure 6B), indicating that MUC4 loci preferentially locate at the nuclear periphery.

69. The human β-globin (HBB) gene, which encodes a subunit of the adult hemoglobin and is mutated in β-thalassemia (Hill et al., 1962). In China, CD14/15, CD17, and CD41/42, which are frame-shift or truncated mutations of β-globin, are three of the most common β-thalassemia mutations (Cao and Galanello, 2010). Located on chromosome 11, HBB is within the β-globin gene cluster that contains four other globin genes with the order of (from 5′ to 3′) HBE, HBG2, HBG1, HBD, and HBB (Schechter, 2008). Because the sequences of HBB and HBD are very similar, HBD may also be used as a template to repair HBB. The HBD footprints left in the repaired HBB locus should enable us to investigate whether and how endogenous homologous sequences may be utilized as HDR templates, information that will prove invaluable to any future endeavors that may employ CRISPR/Cas9 to repair gene loci with repeated sequences.

70. Tried to mutate the human β-globin (HBB) gene in ‘non-viable’ embryos (β-thalassaemia)
7 of 86 embryos were successfully mutated Much more higher rates of off-targeting
Raise huge ethical concerns...

72. Gene Therapy
Programmable nucleases as antivirals. In addition, programmable nucleases may be developed as antiviral therapies.In principle, nucleases may be used to target viral sequences for cleavage and subsequent destruction. Additionally, NHEJ-based mutagenesis of elements critical for viral fitness could render latent viruses incapable of propagating infection. Alternatively, multiplexed nucleases like Cas9 could be used to excise proviruses from the genomes of infected cells, leading to their degradation by cellular nucleases.Efforts to develop genome editing nucleases for antiviral therapy have focused primarily on HIV, where large reservoirs of latent provirus can persist in the presence of anti-retroviral therapies and serve to reactivate infection once treatment is stopped. The long terminal repeats (LTRs) of HIV drive viral gene expression and are critical for viral fitness. One study recently demonstrated the possibility of mutating the proviral LTR by targeting Cas9 to cleave LTR sequences, significantly reducing the expression of HIV genes in T cells64.Although this is an exciting discovery, there are several additional challenges to translating these in vitro results to the clinic. Likely the greatest will be delivering nucleases to all HIV-carrying cells in an infected individual so as to eliminate all of the latent provirus. Currently, there are no therapeutic platforms capable of delivering genome editing nucleases to the majority of T cells. Similar strategies have shown promise with human papillomavirus (HPV)141 and hepatitis B virus (HBV)65,66, but most infectious diseases face the same problem as HIV: extremely efficient delivery of genome editing tools is likely to be needed to achieve complete removal of viral infection

73. Pathogenic mutations can be broadly classified as causing either gain or loss of function in a gene product. A gain-of-function mutation, such as those found in the HTT gene in Huntington disease ( org/entry/143100) and in FGFR3 in achondroplasia ( entry/100800), results in the expression of a pathogenic gene product and may be treated by using NHEJ-mediated mutations to specifically inactivate the mutant allele while leaving the wild-type allele intact on the homologous chromosome (Fig. 1a)
Additionally, it may be pos- sible to treat nucleotide expansion disorders, such as spinocerebellar ataxia ( Huntington disease and Friedriech ataxia ( by NHEJ-based dele- tion of the pathogenic insertion via the creation of two DSBs on both sides of the expansion (Fig. 1b). A combination of DSBs may also be used to edit multiple loci to achieve a therapeutic effect.

74. However, some gain-of-function mutations, such as the SOD1 G93A mutation found in some individuals with amyotrophic lateral sclerosis (ALS) ( are point mutations, which may not be sufficiently different from the functioning allele on the homologous chromosome to be distinguished by the current generation of pro- grammable nucleases, potentially leading to an undesirable complete loss of protein function if the mutation is targeted using NHEJ. In such cases HDR could instead be used to change the gain-of-function allele to the wild-type sequence, restoring gene function and eliminating patho- genic activity while preserving physiological levels of gene expression
For deleterious loss-of-function mutations and protective gain- of-function mutations, a therapeutic effect may also be achieved by introducing a copy of the wild-type gene or gain-of-function mutant, respectively (Fig. 1d).

76. Figure 3Ex vivo versus in vivo editing therapy
Figure 3Ex vivo versus in vivo editing therapy. Top: in ex vivo editing therapy, cells are removed from a patient being treated, edited and then re-engrafted. For this mode of therapy to be successful, the target cells must be capable of surviving outside the body and homing back to target tissues after transplantation. Bottom: in vivo therapy involves genome editing of cells in situ. For in vivo systemic therapy (left), delivery agents that are relatively agnostic to cell identity or state would be used to effect editing in a wide range of tissue types. Alternatively, targeted in vivo therapy may also be achieved through targeted local injection (right) of viral vectors to the affected tissue or through the systemic injection of viral vectors with inherent tropism for specific diseased tissues, such as the eye brain, or muscle.