The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. Additionally, the simple gene targeting mechanism of CRISPR technology has been modified and adapted to other applications that include gene regulation, detection of intercellular trafficking, and pathogen detection. With a wealth of methods for introducing Cas9 and gRNAs into cells, it can be challenging to decide where to start. In this presentation, Dr Adam Clore describes the CRISPR mechanism and some of the most prominent uses for CRISPR, along with methods where IDT technologies can assist scientists in designing, testing, and executing a variety of CRISPR-mediated experiments. For more informaton, visit: http://www.idtdna.com/crispr

1. Rewriting the Genome With gBlocks® Gene Fragments
Harnessing the Power of CRISPR and Synthetic Biology
Adam Clore, PhD
Manager, Synthetic Biology
Design and Support

2. The design, redesign, and construction of new biological
parts, devices, and systems
Defining Synthetic Biology

3. Genome Modification Using the Repair of Double Stranded Breaks
• In most eukaryotes non homologous end joining (NHEJ) is the most efficient
DBS repair pathway
• Error prone, often creates INDELs
• The presence of homologous template (aka “Donor DNA”) can induce
recombination
• Efficiency and length of homology arms varies from one cell line to another

4. Recent Evolution of Genome Editing
• Zinc Finger Nucleases (ZNFs)
• TALENs
• Meganucleases
• CRISPR/Cas9
Curtin (2012), Plant Gen., 5, p42-50

5. Overview of Three Generations of Programmable Nucleases
Cost Reliability Accuracy
Zinc Finger
Nucleases
$$$$ Low Poor
TALENs $$$$ High Good
CRISPR $ High Good

6. CRISPR—Easy Genome Modification
• Clustered Regularly Interspaced Short Palindromic Repeat
• A prokaryotic defense mechanism that screens for and cleaves specific DNA
sequences
• Can be used to create targeted changes to the genomes of bacteria, archaea, and
eukaryotes

7. The 3 Stages of CRISPR Resistance
● Stage 1: CRISPR Adaptation
– Foreign DNA is incorporated in the CRISPR array.
● Stage 2: CRISPR Expression
– CRISPR RNAs (crRNAs) are transcribed from the
CRISPR locus.
● Stage 3: CRISPR Interference
– Foreign nucleic acid complementary to the crRNA
is neutralized.

8. CRISPR Applications
New England Biolabs

9. How Do You CRISPR?
CRISPR System Features
Dual-expression plasmids
Cas9 under constitutive promoter
gRNA under Pol III promoter (U6/H1)
Most common, most published data
Large plasmids (8-10 kb), tricky transfection
Cloning of gRNAs is cumbersome
Two single-expression plasmids Smaller plasmids
Easier cloning of gRNAs
Lower transfection rates
Lentiviral transfection High efficiency
Requires cloning and pseudovirus production
Microinjection Preferred for embryos
Highest efficiency (>95% of cells express Cas9)
Requires specialized equipment and methods
Cell lines expressing Cas9 Eliminates variation in large plasmid transfection
Alternative delivery of CRISPR components
gRNA expression cassette (i.e., gBlocks® Gene Fragments)
gRNAs
Cas9 protein
Alternative PAMs
In search of increased targeting efficiency and
reduced off-target effects

10. CRISPR Mediated Gene Disruption
• CHO cells
• In zebrafish
• In yeast
• The list goes on…

11. CRISPRi
CRISPR Based Gene Silencing

12. Method
• CRISPRi uses a nuclease dead Cas9 Protein to sterically block transcription
elongation or promoter binding
• First demonstrated in E. coli by Qi et al (Weissman Lab, Berkeley) in 2013
http://dx.doi.org/10.1016/j.cell.2013.02.022

13. Transcription Repression

14. Benefits of CRISPRi
• Functions in all domains of life
 siRNA is not active in prokaryotes and some fungi
 No permanent change to genome
 Different activity than RNAi
 Anecdotal information suggests that CRISPR may be more robust that RNAi

15. Screening With CRISPR Libraries
• First large screen done by
Zhang Lab in 2014
• Created genome-scale
CRISPR knockout library
(GeCKO)
• Lentiviral vector with CRISPR
cassette
• Identification of genes
associated with vemurafenib
sensitivity (B-Raf inhibitor)
 18,080 genes
 64,751 gRNAs
Science (2014), 343

16. Screening With CRISPR Libraries
• First large screen done by
Zhang Lab in 2014
• Created genome-scale
CRISPR knockout library
(GeCKO)
• Lentiviral vector with CRISPR
cassette
• Identification of genes
associated with vemurafenib
sensitivity (B-Raf inhibitor)
 18,080 genes
 64,751 gRNAs

17. CRISPR as a Biological Sensor
• Paris-Bettencourt iGEM team
• 1st place in 2013 “overgrad” competition
• Detection of tuberculosis drug resistance genes using a
phage-delivered cassette containing:
• a Cas9/gRNA targeting a drug resistance gene
• a LacZ gene driven by an SOS dependent promoter

18. • Designed for a quick and inexpensive field diagnostic
CRISPR as a Biological Sensor

19. Creating Long Accurate Synthetic DNA Without Cloning
 IDT introduced the concept of synthetic gene fragments
 125–2000 bp in length
 Sequence-verified
 Short delivery time and low price
 200 ng provided, dry
 High quality DNA fragments,
 Fast–assembly and cloning required
Top 3 questions:
- Can you make them longer?
- Can you make them variable?
- Can I get a discount?

20. Using gBlocks® Gene Fragments for CRISPR
www.idtdna.com/gblocks Current Protocols in Molecular Biology (2014), 31.1.1-31.1.17.

21. Using gBlocks® Gene Fragments for CRISPR
www.idtdna.com/gblocks Current Protocols in Molecular Biology (2014), 31.1.1-31.1.17.

22. gBlocks® Gene Fragments for CRISPR

23. Design of Donor DNA
• dsDNA typically requires homology arms >500 bp in mammalian cells
 Caution! HR efficiency and optimal arm length varies greatly between cell lines and must be
experimentally verified
• ssDNA can efficiently recombine with 40–50 base homology arms
TARGET GENE
5’ Arm URA3 3’ Arm

24. gBlocks® Gene Fragments—2015
 We made them longer—up to 2 kb
 We made them variable
 >50 citations
gBlocks® Gene
Fragments
Usually
Shipped (BD) Pricing
125–500 bp 2–4 $89.00 USD
501–750 bp 2–4 $129.00 USD
751–1000 bp 3–5 $149.00 USD
1001–1250 bp 5–8 $209.00 USD
1251–1500 bp 5–8 $249.00 USD
1501–1750 bp 5–8 $289.00 USD
1751–2000 bp 5–8 $329.00 USD

25. Surveyor® Detection of CRISPR Modifications
(B) sgRNA + CAS9 bind and cut their target sequence creating a
double-strand break (DSB) in a portion of the cells.
(C) Aberrant repair of some DSBs by non-homologous end
joining (NHEJ) results in insertion, deletion or substitution
(depicted by red X).
(A) CRISPR sgRNA delivered to cells.
sgRNA +
Cas9

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Surveyor® Detection of CRISPR Modifications
(C, D) Genomic DNA is harvested from the transfected
pool of cells and amplified at the locus of interest.
(E, F) PCR product is denatured and re-annealed creating
heteroduplex formation between wild type and modified
amplicons.

27. Quantitative Assessment of CRISPR Gene Editing via Mismatch Endonuclease
Average % Cleavage of Biological Triplicates via Fragment Analyzer™

29. Biosecurity
• IDT is one of the five founding members of the International Gene Synthesis
Consortium (IGSC)
• Screens the sequence of every gene and gBlocks® Gene Fragment order
• To ensure safety and regulatory conformance
• IDT reserves the right to refuse any order that does not pass this analysis
• For more information about the IGSC and the Harmonized Screening Protocol, please visit the
website at http://www.genesynthesisconsortium.org/Home.html.
• In October of 2010, the United States government issued final Screening Framework Guidance for
Providers of Synthetic Double-Stranded DNA, describing how commercial providers of synthetic
genes should perform gene sequence and customer screening. IDT and the other IGSC member
companies supported the adoption of the Screening Framework Guidance, and IDT follows that
Guidance in its application of the Harmonized Screening Protocol. For more information, please see
75 FR 62820 (Oct. 13, 2010), or http://federalregister.gov/a/2010-25728.

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CRISPR Resources
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• www.IDTDNA.com/gBlocks
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Issues, and Ordering
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