1. CRISPR and Targeted Genome Editing
(CRISPR: Clustered Regularly Interspaced Short Palindromic
Repeats)
Dr Nemat Sokhandan Bashir
Associate Professor
University of Tabriz

2. Current Circumstance Lots of sequencing data are available
Relating of these data to phenotype is a major challenge
Reverse genetics
Forward genetics

3. Protein-based Approaches
Protein directed
Recombinases
Integrases
ZFNs (zinc finger nucleases)
TALENs (transcription-activator-like effector nucleases)

4. Shortcomes of Protein-based Approaches
Harder to customize
Recombinases and integrases require suitable preexisting recognition sites in the genome
-often have some inherent application limitations
Difficult and expensive to customize ZFs or TALs by protein engineering
ZFN and TALEN activities are affected by many factors
ZFN and TALEN difficult for multiple mutations in a single genome

5. Nucleotide-based Approaches
RNA interference (RNAi),
group II intron retrotransposition,
widely applied to inactivate genes in bacteria
Cas9-based platforms (CRISPR)

6. RNAi Need for long target sites
Amplification of small RNAi -severe off-target effects
Repress gene expression instead of knocking them out

7. History of CRISPR First noticed in bacterial genomes (1987)
Japanese examining E. coli iap gene
Could not attach biological significance to such “unusual structure[s]”
Nearly a decade later, a group from Spain recognized that these repeating sequences were a common feature in microbial genomes.

8. characteristic of CRISPR
A series of palindromic DNA repeats (20-50 bp)
separated by spacer sequences of ~ 20-50
Precise function (2005):
spacer sequences were often consistent with nt sequences found in phages
suggesting a role in bacterial innate immunity

9. characteristic of CRISPR
2007: resistance to a phage could be altered by modifying bacterial spacer DNA
Spacers between palindromic repeats correspond to phage DNA sequences
Spacers as “memory” of prior infection
“memory” alone is not enough to protect bacteria from subsequent exposure to phage

10. exposure to a previously-exposed phage -> targeted DNA-cutting function
Triggers transcription of the corresponding spacer sequence into a short guide CRISPR-RNA (crRNA)
The CRISPR-associated (“Cas”) enzyme (Cas-9) is then guided to the target phage by a
two-part RNA structure bears a short region of homology to the original phage DNA.
Cas-9 identify and cleave the phage DNA, creating a dsDNA cut at a specific site.

11. Classification of CRISPR/Cas system
On sequence and structure of Cas protein
Types I, II, and III
crRNA-guided surveillance complexes in types I and III need multiple Cas subunits
Type II requires only Cas9

12. CRISPR/Cas type II Studied in Streptococcus and Neisseria
Distinct in each species
pre-crRNA transcript features
pre-crRNA processing
nucleoprotein complexes
A promising programmable tool
three crucial components:
RNA-guided Cas9 nuclease
crRNA (CRISPR RNA)
a partially complementary trans-acting crRNA (tracrRNA)

13. crRNA biogenesis pre-crRNA processing and length varies in species
Streptococcus pyogenes produces only one form of full-length primary pre-crRNA of 511 nt, consisting a leader region and a number of repeat-spacer-repeat units

14. tracrRNA
a non-protein-coding RNA for crRNA maturation and subsequent DNA cleavage
In S. pyogenes, the tracrRNA gene is transcribed from two start sites producing two primary species of 171 nt and 89 nt, both are processed into75-nt RNA species
tracrRNA precursors have almost perfect (one mismatch) complementarity with each of the pre-crRNA repeats.
Base-pairing RNA duplex is important for tracrRNA
precursor trimming and crRNA maturation

16. CRISPR/Cas-based method highly flexible and programmable
All of the essential components can be expressed by delivering
Plasmids
linear DNA expression cassettes
RNA transcripts
most genes or exons can be targeted specifically in Arabidopsis, rice and yeast ((N21GG))

17. Genomic target can be any ∼20 nucleotide DNA, if:
Sequence is unique compared to the rest of the genome.
Target is present immediately upstream of a Protospacer Adjacent Motif (PAM).

18. Cas9 nuclease (formerly Csn1 or Csx12)
Cleaves dsDNA (sequence-specific)
RuvC-like nuclease domain at N-terminus
Named for an E. coli DNA repair protein
HNH (or McrA-like) nuclease domain in the middle
Named for histidine and asparagine residues
Each cuts opposite DNA strand to give DSBs

20. Generating a Knock-out Using CRISPR/Cas9
S. pyogenes Cas9 most widely used
Once expressed, Cas9 protein and gRNA form a riboprotein complex through:
Interactions between the gRNA “scaffold” domain and surface-exposed positively-charged grooves on Cas9
Cas9 undergoes a conformational change
Shifts from an inactive, non-DNA binding conformation, into an active DNA-binding conformation
“spacer” sequence of the gRNA remains free to interact with target DNA

24. DSB is repaired by one of two general repair pathways
The efficient but error-prone Non-Homologous End Joining (NHEJ) pathway
The less efficient but high-fidelity Homology Directed Repair (HDR) pathway

25. NHEJ repair The most active repair mechanism of DSBs
But frequently gives small nt InDels at DSB site
This randomness has practical implications:
because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations

26. NHEJ repair Mostly results in small InDels in the target DNA
in-frame amino acid deletions, insertions, or
frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene.
Ideally, the end result is a loss-of-function mutation within the targeted gene;

27. Increase specificity More specific gRNA with no off-targets
dual nickase approach

28. dual nickase approach for more specificity
Cas9 generates DSBs by combined activity of RuvC and HNH.
Exact AA residues within each domain critical for endonuclease activity are known
D10A for HNH and H840A for RuvC in S. pyogenes Cas9
modified versions of the Cas9 enzyme containing only one active catalytic domain (“Cas9 nickase”)

29. Cas9 nickases Still bind DNA based on gRNA specificity,
Cuts only one strand
Nicks are rapidly repaired by HDR
Two nickases targeting opposite strands are required
Unlikely that two off-target nicks will be generated within close enough proximity to cause a DSB.
The nickase system can also be combined with HDR-mediated gene editing for highly specific gene edits.

30. Increasing efficiency of HDR
HDR efficiency is generally low (<10% of modified alleles)
synchronizing the cells within the cell cycle stage when HDR is most active, or
by chemically or genetically inhibiting genes involved in NHEJ

31. implications of low efficiency of HDR
efficiency of Cas9 cleavage is relatively high
efficiency of HDR is relatively low,
a portion of the Cas9-induced DSBs will be repaired via NHEJ.
In other words, resulting cell population will contain: combination of wild-type alleles, NHEJ-repaired alleles, and/or the desired HDR-edited allele

33. Applications

34. Know your cell line and genome sequence
Select gene and genetic element to be manipulated
Select gRNAs based on predicted “on-target” and “off-target” activity
Synthesize and clone desired gRNAs
Deliver Cas9 and gRNA
Validate genetic modification

36. An Example of Genome Editing Assay

37. a chromosomal deletion by targeting two adjacent sequences

38. Targeting virus

39. Pooled Lentivirus CRISPR libraries

40. CRISPR/Cas9 and Plant Viruses
Plant viruses can deliver CRISPR/Cas9 components
Manipulation and improvement of R gene
Rx of potato is a fast HR- a potential use

41. Purification of genomic region by Chromatin immunoprecipitation

42. Cas9-based Forward Genetic Screen

44. Fungi to produce diverse enzymes or metabolites
Trichoderma reesei,
Aspergillus niger,
A. oryzae,
Penicillium chrysogenum,

45. To obtain hyper-producers
classical mutagenesis
Genetic engineering well developed
These are not efficient due to:
additional complexity such as multicellular morphology, cellular differentiation, thick chitinous cell walls, and the lack of suitable plasmids

46. Prospects and Challenges
Discover new gene functions with high sensitivity and precision
1000s of guides target all coding genes in genome genome-scale gain- and loss of function genetic screens
Identify novel disease-protective mutations such as loss-of-function mutations in CCR5 protection to HIV
Direct treatment of genetic diseases via genome editing of somatic cells

47. Treatment of eye and hearing disorders actively evaluated in animal models
Many groups striving to make CRISPR/Cas9-based therapies a reality
Raises certain societal challenges and brings a sense of uncertainty and fear of catastrophic misuse
One thing is certain—nature will never cease to inspire us with its biological toolbox
The tools themselves do not pose a threat
Lets hope CRISPR/Cas9 technology will live up to its promise by being used responsibly and carefully

48. References
CRISPR Guide, Addgene, Accessed 7 Decebmer 2015.
Xu,T., Li,L., D. Van Nostrand,J., He,Z., Zhou, J Cas9-Based Tools for Targeted Genome Editing and Transcriptional Control. Applied and Environmental Microbiology, 80(5):1544–1552.
Peng, C., Lu, M., Yang, D CRISPR/Cas9-based tools for targeted genome editing and replication control of HBV.
VIROLOGICA SINICA 2015, 30 (5):
Feng, Z. CRISPR/Cas9: Prospects and Challenges. HUMAN GENE THERAPY, 26(&):
Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Nekrasov, V Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Belhaj et al. Plant Methods, 9:39.