Garrett Rettig PhD, Research ScientistIntegrated DNA Technologies

Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Design and use

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Implementing CRISPR-Cas9 gene editing

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Complexed RNA oligonucleotides as the CRISPR gRNA

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Optimal crRNA length is 36 nt; optimal tracrRNA is 67 nt

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Alt-R™ CRISPR-Cas9 System products from IDT

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Description Formats

Alt-­‐R  CRISPR  crRNA Tubes:  2,  10  nmol;  Plates:  2  nmol

Alt-­‐R  CRISPR  tracrRNA 5,  20,  100  nmol

Alt-­‐R  HPRT  Positive  Control  Kits 2  nmol:  human,  rat,  mouse

Alt-­‐R  HPRT  Positive  Control  crRNA 2  nmol:  human,  rat,  mouse

Alt-­‐R  Negative  Control  crRNA 3  sequence  options

Alt-­‐R  HPRT  PCR  Primer  Mix 2  nmol  (each):  human,  rat,  mouse

Alt-­‐R  S.p.  Cas9  Expression  Plasmid Minimal  plasmid  (7  kb)

Before you begin

• Considerations for the crRNA design– Genomic DNA target– Exon for protein disruption– Non-coding genomic region for HDR– PAM sites and crRNA in region of interest

• Planning your Alt-R CRISPR-Cas9 System editing experiment– Cell line and tissue culture reagents– Cas9 expression via Alt-R S.p. Cas9 Expression

Plasmid– Alt-R CRISPR RNAs– Reagents for downstream detection of edits

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Most Alt-R CRISPR crRNAs provide good to excellent editing performance, minimizing the need for extensive design• General rules for on-target

– Target coding regions or exon/intron junctions

– Avoid 5′ and 3′ UTR– Moving towards the C-terminus reduces

hit rate– Exon > 1 kb at 5′ end of the gene

General rules for designing an Alt-R™ CRISPR crRNA

7Nat  Biotechnol,  32(12):  1262–1267  

We currently suggest using the free CRISPR design tool at http://crispr.mit.edu/• Input genomic DNA target sequence

– 250 bases of your dsDNA target– 16 unique species reference genomes– Batch entry option—parallel search

• Scoring readout: 0.0–1.0– Hsu, et al 2013, Nature Biotechnology– Mismatch position/distance between– Bioinformatically eliminate numerous

off-targets

Check your crRNA for potential off-target effects

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Results from the design tool at http://crispr.mit.edu/• From the genomic DNA input, every PAM site and associated protospacer (top

and bottom strand) is annotated and scored.

Selecting a crRNA with reduced off-target effects

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Results from the design tool at http://crispr.mit.edu/• Green indicates minimal predicted off-target effects• Yellow indicates guides should only be used when better alternatives are not available• Red indicates that these guide RNAs should be avoided

Selecting a crRNA with reduced off-target effects

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All PAM sites in 6 exons, 553 sites (HEK293-Cas9 cells)* Percentage of crRNA designs with >20 % editing efficiency by T7EI assay

Observed performance of Alt-R™ CRISPR crRNAs

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* * *

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• The guide RNA is identical to the DNA sequence 5′ of the PAM• The guide RNA is complementary to the strand opposite the PAM site• Input only the 20 or 19 nt target sequence. Remaining 3′ RNA sequence is added

automatically

Ordering the correct crRNA sequence

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• Enter either 19 or 20 nt spacer sequence– 16 nt will automatically be added to

complete your 35 or 36 nt crRNA• Consider crRNA position relative to PCR assay

design (or other editing detection method)• GC content of sense and antisense strand

– No impact on function– Relevant for the number of NGG PAM sites,

and PAM site orientation• Scale: 2 and 10 nmol• Batch entry option, 2 nmol plate orders

– Minimum order of 24 crRNAs

Ordering Alt-R™ CRISPR crRNAs

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Begin your order at www.idtdna.com/CRISPR

Ordering individual Alt-R™ CRISPR crRNAs

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Begin your plate order at www.idtdna.com/CRISPR• Minimum order for

plates is 24 crRNAs

• 2 nmol scale

Ordering Alt-R™ CRISPR crRNAs in plates

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The Alt-R™ CRISPR tracrRNA is required for complexing with the Alt-R™ CRISPR crRNA• Following your crRNA entry you will be prompted to order the tracrRNA, and other

optional controls and reagents

Ordering Alt-R™ CRISPR tracrRNA

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Available individually, or in kits, for human, mouse, and rat

• Kits include: HPRT crRNA, PCR primers, tracrRNA, and negative control crRNA

Ordering optional Alt-R™ CRISPR Control crRNAs

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• Smaller than other Cas9 plasmids at only 7.3 kb

• Editing efficiency improved over other commercially available plasmid

• 1 µg of dry plasmid is supplied, you will need to transform competent E. coli and generate additional plasmid for experiments

Ordering Alt-R™ S.p. Cas9 Expression Plasmid

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Complete experimental instructions can be found in the User Guide PDF at www.idtdna.com/CRISPR, under the support tab

Experimental outline for the Alt-R™ CRISPR-Cas9 System

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Using the Alt-R™ S.p. Cas9 Expression Plasmid

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Genome editing using the Alt-R™ CRISPR-Cas9 System

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1. PCR amplify region flanking the CRISPR site (400–1000 base amplicons)– Heat, cool to form heteroduplexes

2. Incubate with T7EI (New England BioLabs) 3. Run on gel or Fragment Analyzer™ (Advanced Analytical) to visualize cleavage at heteroduplex

mismatch sites

Verify editing using T7 Endonuclease I (T7EI) assay

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• Design primers for your target site at www.idtdna.com/PrimerQuest– We recommend that you generate

a 600–1000 bp PCR amplicon with >100 bp flanking the CRISPR cut site.

• Order Alt-R™ HPRT Positive Control Primer Mix for human, mouse, or rat positive controls at www.idtdna.com/CRISPR

Verify editing using T7EI assay

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Workflow for evaluation of CRISPR events using T7EI

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Complex  crRNA  and  tracrRNA  in  buffer;  95°C  for  2  min

Transfect  complexed  RNAs  at  

30  nM

Extract  gDNA after  48  hr with  

QuickExtract™  DNA  Solution

Heat  gDNA extract  at  65°C  for  15  min  followed  by  95°C  

for  15  min

Amplify  gDNA with  KAPA  HiFi

Polymerase  and  PCR  assay  targeting  region  of  interest

Add  NEB  buffer  2  to  PCR,  heat  to  95°C  and  slowly  cool  to  allow  heteroduplex  

formation

Digest  heteroduplexes  

with  2  units  of  T7EI  at  37°C  for  1  hr

Analyze  digestion  on  Fragment  Analyzer™

The Fragment Analyzer™ (Advanced Analytical) provides reliable quantification of T7EI heteroduplex cleavage assay with 96-channel CE

– High resolution analysis of fragments 10–40,000 bp– Rapid 1 hr run – 1/10th amount of DNA required to visualize

Visit www.idtdna.com/CRISPR for more information• Support

– User Guide and short protocols– Previous webinar on Alt-R™ CRISPR-

Cas9 System data– Short tutorial videos on how to

order• Performance

– View key data from Alt-R™ CRISPR-Cas9 System experiments

Alt-R™ CRISPR-Cas9 System additional resources

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Questions?

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