CRISPR and Targeted Genome

Editing
(CRISPR: Clustered Regularly
Interspaced Short Palindromic
Repeats)
Dr Nemat Sokhandan Bashir
Associate Professor
University of Tabriz
 Current Circumstance
• Lots of sequencing data are available
• Relating of these data to phenotype is a major
challenge
– Reverse genetics
– Forward genetics
 Protein-based Approaches
• Protein directed
• Recombinases
• Integrases
• ZFNs (zinc finger nucleases)
• TALENs (transcription-activator-like effector
nucleases)
 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
 Nucleotide-based Approaches
• RNA interference (RNAi),
• group II intron retrotransposition,
– widely applied to inactivate genes in bacteria
• Cas9-based platforms (CRISPR)
 RNAi
• Need for long target sites
• Amplification of small RNAi -severe off-
target effects
• Repress gene expression instead of knocking
them out
 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.
 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
 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
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.
 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

 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)
 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
 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
 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))
 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).
 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
 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
 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
 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
 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;
 Increase specificity
• More specific gRNA with no off-targets
• dual nickase approach
 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”)
 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.
 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
 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
Applications
• 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
 An Example of
Genome Editing
Assay
a chromosomal deletion by targeting two adjacent sequences
Targeting virus
Pooled Lentivirus
CRISPR libraries
 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
Purification of genomic
region by Chromatin
immunoprecipitation
Cas9-based Forward Genetic Screen
 Fungi to produce diverse
enzymes or metabolites
• Trichoderma reesei,
• Aspergillus niger,
• A. oryzae,
• Penicillium chrysogenum,
 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
 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
• 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
 References
• CRISPR Guide, Addgene,https://www.addgene.org/crispr/guide/. Accessed 7
Decebmer 2015.
• Xu,T., Li,L., D. Van Nostrand,J., He,Z., Zhou, J. 2014. Cas9-Based Tools for
Targeted Genome Editing and Transcriptional Control. Applied and
Environmental Microbiology, 80(5):1544–1552.
• Peng, C., Lu, M., Yang, D. 2015. CRISPR/Cas9-based tools for targeted genome
editing and replication control of HBV.
• VIROLOGICA SINICA 2015, 30 (5): 317-325
• Feng, Z. CRISPR/Cas9: Prospects and Challenges. HUMAN GENE THERAPY,
26(&): 409-410.
• Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Nekrasov, V. 2013. Plant genome
editing made easy: targeted mutagenesis in model and crop plants using the
CRISPR/Cas system. Belhaj et al. Plant Methods, 9:39.