Rewriting the Genome With gBlocks® Gene Fragments

Harnessing the Power of CRISPR and Synthetic Biology

Adam Clore, PhD

Manager, Synthetic Biology

Design and Support

The design, redesign, and construction of new biological parts, devices, and systems

Defining Synthetic Biology

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

Recent Evolution of Genome Editing

• Zinc Finger Nucleases (ZNFs)

• TALENs

• Meganucleases

• CRISPR/Cas9

Curtin (2012), Plant Gen., 5, p42-50

Overview of Three Generations of Programmable Nucleases

Cost Reliability Accuracy

Zinc Finger Nucleases

$$$$ Low Poor

TALENs $$$$ High Good

CRISPR $ High Good

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

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.

CRISPR Applications

New England Biolabs

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 componentsgRNA expression cassette (i.e., gBlocks® Gene Fragments)

gRNAs

Cas9 protein

Alternative PAMs

In search of increased targeting efficiency and

reduced off-target effects

CRISPR Mediated Gene Disruption

• CHO cells

• In zebrafish

• In yeast

• The list goes on…

CRISPRi

CRISPR Based Gene Silencing

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

Transcription Repression

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

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

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

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

• Designed for a quick and inexpensive field diagnostic

CRISPR as a Biological Sensor

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?

Using gBlocks® Gene Fragments for CRISPR

www.idtdna.com/gblocks Current Protocols in Molecular Biology (2014), 31.1.1-31.1.17.

Using gBlocks® Gene Fragments for CRISPR

www.idtdna.com/gblocks Current Protocols in Molecular Biology (2014), 31.1.1-31.1.17.

gBlocks® Gene Fragments for CRISPR

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

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

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.

Quantitative Assessment of CRISPR Gene Editing via Mismatch Endonuclease

Average % Cleavage of Biological Triplicates via Fragment Analyzer™

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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INTEGRATED DNA TECHNOLOGIES

Additional ResourcesCRISPR Resources

• www.IDTDNA.com/CRISPR

Information for gBlocks® Gene Fragments

• www.IDTDNA.com/gBlocks

Support for Help With Design, Experimental

Issues, and Ordering

• Genes@IDTDNA.com

Other Educational Resources at www.IDTDNA.com

Under Support & Education Menu:

• DECODED Newsletter

(www.IDTDNA.com/DECODED)

• Video Library

• Frequently Asked Questions

• More…

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