Tools and Resources

This is an area for fellow researchers to share tools and websites that they have found useful in their genome engineering projects.  If you feel you have a useful addition (or a correction) please email us at the contact email found at the bottom of the page.

Websites, Reviews, Target Finder Tools, Discussion Groups

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Review Articles

CRISPR/Cas9 and genome editing in Drosophila.
Bassett AR, Liu JL.
J Genet Genomics. 2014 Jan 20;41(1):7-19. doi: 10.1016/j.jgg.2013.12.004. Epub 2013 Dec 18.
PMID: 24480743
CRISPR/Cas9 mediated genome engineering in Drosophila.
Bassett A, Liu JL.
Methods. 2014 Feb 24. pii: S1046-2023(14)00063-2. doi: 10.1016/j.ymeth.2014.02.019. [Epub ahead of print]
PMID: 24576617
Targeted genome engineering techniques in Drosophila.
Beumer KJ, Carroll D.
Methods. 2014 Jan 8. pii: S1046-2023(13)00455-6. doi: 10.1016/j.ymeth.2013.12.002. [Epub ahead of print]
PMID: 24412316
Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes.
Copeland MF, Politz MC, Pfleger BF.
Curr Opin Biotechnol. 2014 Mar 12;29C:46-54. doi: 10.1016/j.copbio.2014.02.010. [Epub ahead of print] Review.
PMID: 24632195
CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand.
Gratz SJ, Wildonger J, Harrison MM, O’Connor-Giles KM.
Fly (Austin). 2013 Oct 2;7(4). [Epub ahead of print] Review.
PMID: 24088745
ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.
Gaj T, Gersbach CA, Barbas CF 3rd.
Trends Biotechnol. 2013 Jul;31(7):397-405. doi: 10.1016/j.tibtech.2013.04.004. Epub 2013 May 9. Review.
PMID: 23664777
Function and regulation of clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated (Cas) systems.
Richter C, Chang JT, Fineran PC.
Viruses. 2012 Oct 19;4(10):2291-311. doi: 10.3390/v4102291. Review.
PMID: 23202464
RNA-guided genetic silencing systems in bacteria and archaea.
Wiedenheft B, Sternberg SH, Doudna JA.
Nature. 2012 Feb 15;482(7385):331-8. doi: 10.1038/nature10886. Review.
PMID: 22337052
CRISPR/Cas, the immune system of bacteria and archaea.
Horvath P, Barrangou R.
Science. 2010 Jan 8;327(5962):167-70. doi: 10.1126/science.1179555. Review.
PMID: 20056882

Target Finding Tools

CRISPR Optimal Target Finder – (flyCRISPR, University of Wisconsin at Madison)

E-CRISP – (Heigwer F. Nat Methods. 2014, German Cancer Research Center)

DRSC Find CRISPRs – (Perrimon Lab, Harvard Medical School)

CRISPR Design and Off Target Effects – (Zhang Lab, MIT)

CasOT Off Target Effects Tool – (Peking University)

Discussion Groups


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Getting Started

What is CRISPR?

CRISPRs (clustered regularly interspaced short palindromic repeats) are repetitive nucleotide sequences followed by a short spacer DNA segments integrated into the genomes of many bacteria and archaea upon exposure to invading pathogens.  CRISPR-associated (Cas) nucleases form the CRISPR-Cas machinery to rid the prokaryotes of foreign genomic elements.  In 2012, CRISPR was utilized in eukaryotes and a readily-programmable two-component CRISPR system was used to disrupt targeted genomic sites. In the short time since, many genome engineering strategies have been developed that would have previously been laborious and costly (many of these developments were made here at UW, including Drosophila and stem cell work).

What can I do with CRISPR?

CRISPR-Cas can make genetic manipulations, great, couldn’t we already do that?

The CRISPR-Cas system is similar to other efforts to modify the genome such as the ZFNs (Zinc Finger Nucleases) and TALENs (Transcription activator-like effector nucleases), however, it differs from these techniques in its simplicity.  This simplicity, unfortunately, has also lead to some specificity issues that are currently being investigated and will be discussed below.

The CRISPR-Cas system can be used to make:

  • permanent gene deletion in cell lines similar to, but more complete than, RNAi (ref)
  • genomic knockouts of genes in mice (and other model organisms) in less than 1/3 the normal time (ref)
  • genomic deletion of enhancers that drive gene transcription in full organisms or cell lines (ref)
  • targeted genomic knock-in of reporter and resistance genes or LoxP sites for tissue selective removal (ref)
  • disease related genetic mutation correction through targeted homology directed recombination (ref)

The list of what CRISPR can do is growing almost daily and represents one of the most rapidly growing techniques in genetic research today.

Double Strand Breaks, Nicks, or Transcriptional Blockade?

Double Strand Break

By default, the wild type Cas9 enzyme will perform a double strand break.  This double strand break is then repaired by the cell machinery through non-homologous end joining (NHEJ).  Alternatively, if there is a homologous DNA sequence template that is present in the cell, the area may then be repaired by using that template for its repair in a process called Homology Directed Repair (HDR).  This template could be a natural piece of DNA in the cell that shares a sequence similarity, a transposon like region, or this template could be foreign DNA that is introduced into the cell.  Overall, the double strand break version of the CRISPR-Cas system is very efficient when introduced into the cell.  However, double strand breaks are more toxic to the cell as they may not always be repaired efficiently.  This may be overcome by the “Nickase” version of the enzyme.

Nickase Single Mutation Cas Enzyme

It was discovered that a single point mutation in the Cas9 enzyme will result in only one strand of the DNA becoming “nicked” instead of a double strand break occurring.  This DNA nick is more easily repaired by the cell and may be less toxic than a double strand break.  Research suggests that a DNA nick is more conducive to HDR than the double strand break (Cong L. Science. 2013) of the human codon optimized Streptococcus pyogenesCas system of genome engineering, as nicks may be more easily repaired and repaired more seamlessly than a double strand break.  Moreover, it was discovered that two nicks on opposite strands of DNA in close enough proximity would cause a double strand break (Ran FA. Cell. 2013).  This may in fact further the specificity of the CRISPR-Cas system.

Transcriptional Blockade by Double Mutation Cas9

The final version of the hSpCas9 that was investigated was one in which both the D10A and H840A residues were mutated.  This results in an enzymatically dead Cas9 that is unable to induce double strand breaks or single strand nicks.  However, the CRISPR-Cas9 system is still able to locate its target sequence in DNA.  This may be advantageous to interfere with proteins that may normally bind to that region of the genome, such as near gene promoters and transcriptional machinery.  This double mutant form of Cas9, dubbed CRISPRi, has been successfully shown to block transcription in bacteria (Qi LS. Cell. 2013) and eukaryotes (Gilbert LA. Cell. 2013).

Design Tools and Off-Target Effects

Targeting sequences and PAMs

So, what does our actual targeting sequence look like for CRISPR?  As mentioned above, its a simple configuration of a 20bp sequence of DNA followed by a protospacer adjacent motif (PAM) that varies based on source of Cas protein.  It’s this simple configuration leads to its flexibility, but also its specificity issues.  For simplicity we will discuss the Streptococcus pyogenes CRISPR-Cas9 and its PAM recognition motif with is a NGG, where N can be any A, C, G or T base.


To facilitate its transcription in plasmids that have been optimized for this process, the targeting sequence and guide RNA are preceded by the U6 promoter.  This U6 promoter requires that the sequence start with a G for transcription to occur, therefore finding a sequence in which the first base is a G may be advantageous.  However, if no sequence can be found that starts with a G, you can simply append a G on to your sequence in effect making it a 21bp targeting sequence.  This will not affect your targeting efficiency.

This targeting sequence will need to be cloned into one of many available vectors.  This popular vector is from the Zhang Lab at MIT that has done much of the pioneering work on utilizing CRISPR for mammalian engineering.

Here, the target DNA sequence (without the PAM sequence) is cloned into the pX330 plasmid which is available in the Addgene repository.  Cloning information is available on the Zhang Lab website.  Note the appended sequence information for cloning before ordering.  We see that this target sequence is trailed by the remainder of the guide RNA sequence (or 85nt of tracrRNA) and is terminated by a run of 5 Ts (a U6 requirement).

Finally, this plasmid also encodes the hSpCas9 (human codon optimized streptococcus pyogenes Cas9 enzyme) that contains a FLAG tag as well as several nuclear localization signals (NLS) under the CBh promoter control.  Therefore, with one single plasmid, the Cas9 and targeting guide RNA can be delivered in one transfection.

Off-Target Effects

Due to the small sequence recognition for desired targets, there will be some unwanted off-target effects without some careful thought and preparation.  The desired target sequence, including PAM, needs to be compared against to genome to identify all known potential off-target sites.  This, however, is complicated by the fact that it appears the first 12 bases nearest the PAM are the most important for recognition, the remaining 8 bases can have some mismatches (Cong L.Science. 2013).  Therefore to try to combat this bioinformatically, several algorithms and tools have been developed with CRISPR-specific rules.  Below are 3 of the more popular tools for finding optimal targeting sites and minimizing of the off-target effects as much as possible.

CRISPR Design and Off Target Effects – (Zhang Lab, MIT)

CRISPR Optimal Target Finder – (flyCRISPR, University of Wisconsin at Madison)

CasOT Off Target Effects Tool – (Peking University)

It is important to screen the potential targets for off-target possibilities that land within coding exons genes.

Another strategy for limiting the off-target effects would be through usage of closely spaced DNA nicks provided by the mutant forms of Cas9, such as the Cas9-D10A and the Cas9-H840A mutations.  It was discovered that two nicks on opposite strands of DNA in close enough proximity would cause a double strand break (Ran FA. Cell. 2013).  This will increase the specificity of the targeted sequence as the chances decrease dramatically that two off-target nicks will be in as close of a proximity as your targeted sequence.

Plasmids for Consideration

Researchers at UW and elsewhere have been using Addgene for centralized distribution of these CRISPR plasmids.

Addgene CRISPR/Cas Plasmids and Protocols

Also, check out our special Reagents page, only available to those with a UW NetID, which will describe unpublished reagents (including plasmids) developed here at UW. The Reagents page will make these reagents available only to UW labs (coming soon).

Highlighted here are a few of our favorites:

flyCRISPR plasmids for disruptions and homology-directed repair

  • Harrison/O’Connor-Giles/Wildonger lab (UW)
    • pHsp70-Cas9 – The codon-optimized S. pyogenes Cas9 nuclease under the control of the Drosophila hsp70 promoter used in Gratz, et al. (2013).
    • pU6-BbsI-chiRNA – Plasmid for expression of S. pyogenes chiRNA under the control of the Drosophila snRNA:U6:96Ab promoter.
    • pDsRed-attP – Vector for generating dsDNA donors for homology-directed repair to replace genes or other genomic sequence with an attP docking site. Contains the visible marker 3xP3-DsRed. (Also known as pHD-DsRed-att).
    • Information at addgene.
    • Protocols at flyCRISPR.

Neisseria meningitidis CRISPR/Cas9 system for mammalian expression

Single Plasmid Delivery CRISPR Plasmids (guideRNA + S. pyogenes Cas9)

  • Zhang Lab (MIT)
    • pX330 – wildtype Cas9 system
    • pX335 – Cas9-D10A “nickase” system
    • pX458/459 – 3rd generation plasmids with GFP or Puromycin selection (wildtype Cas9)
    • pX461/462 – 3rd generation plasmids with GFP or Puromycin selection (Cas9-D10A)

Two Plasmid Delivery CRISPR Plasmids (guideRNA separate from Cas9)

  • useful for establishing stable cell lines of inducible Cas9 into which the guides can be transfected.
  • Lander/Sabatini Labs (MIT)
    • pCW-Cas9 – doxycycline inducible Cas9 (lentiviral plasmid, puromycin resistant)
    • pLX-sgRNA – blasticidin resistant U6 driven guide RNA (lentiviral plasmid)

Cloning Techniques and Tips

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Gibson Isothermal Assembly

This assembly method for cloning without the use of restriction enzymes was pioneered by Dr. Daniel Gibson at the J. Craig Venter Institute (Gibson DG. Nature Methods. 2009).  It uses homologous sequences to anneal complementary fragments of DNA together, then a polymerase will fill back the gap, and finally a ligase that seals the nicks in the assembled DNA.  The resulting DNA can be directly transformed into your e. coli host of choice for propagation.

The design of Gibson Assembly fragments and primers is quite simple.  You will need to create regions of homologous sequence using primers that span at least 20bp.  Construction and primer design can be aided by building your sequences in a product like SeqBuilder from DNAstar (or any other sequence editor).  Once the desired product is entered into the sequence editor, simply create 5′ and 3′ primers that span 20bp into each backbone and insert.  Do this at each end of the insert.  The resulting 4 primers can be paired to make 2 products (insert and backbone) and PCR amplified using a proofreading Taq like Fusion or PFU and placed into the Gibson Assembly reaction.

As an alternative to PCR, double stranded DNA inserts can be ordered directly from IDT with homologous sequence overhangs.  This may be advantageous if you are cloning in a unique or difficult fragment to PCR.  IDT gBlocks can be synthesized from 120 to 500bp for the same price.  Over 500bp is more expensive.  Tip: backbones can also be enzyme digested linearized fragments.

Gibson Assembly can be used to assembly 2-4 products of varying size quite easily.  To assemble more than 4 products together, it may be more useful to use Ligase Cycling Reaction (LCR), see below.

Diagram of Gibson Assembly from the New England Biolabs Gibson Assembly product page

Gibson Assembly Mix

This Gibson Assembly Master Mix [GA MM] recipe was adapted from the Pfleger Lab and Gibson DG. Nature Methods. 2009publication.

  • T5 Exonuclease [NEB, M0363S (10,000 U/mL) 1,000 units]
  • Phusion High Fidelity DNA Polymerase [NEB, M0530S (2,000 U/mL) 100 units]
  • Taq DNA Ligase [NEB, M0208L (40,000 U/mL) 10,000 units] – [limiting reagent]
  • b-Nicotinamide adenine dinucleotide hydrate (25mg) – [Sigma, N1636-25mg]
  • Poly(ethylene glycol) MW 8000 – [Sigma, P5413-500G]

Gibson Master Mix 5x Isothermal Reaction Buffer (store at -20ºC):

Component Amount
1M Tris-HCl pH 7.5 3 mL
1M MgCl2 300 µL
100mM dNTP 60 µL
PEG-8000 1.5 g
NAD 19.9 mg
1M DTT 300 µL
water 2.22 mL

Gibson Assembly Master Mix [GA MM] (100µL aliquots, store at -20ºC):

Component Amount
5x Isothermal Reaction Buffer 320 µL
T5 exonuclease (10U/µL) 0.64 µL
Phusion DNA Polymerase (2U/µL) 20 µL
Taq DNA Ligase (40U/µL) 160 µL
water 700 µL

Gibson Assembly Reaction

  • PCR amplify your Gibson Assembly products using the above designed primers.
  • If the PCR product is very clean (one singular product), the reaction can DpnI digested for 1 hour followed by clean up with a PCR purification kit (recommended kit is the Zymo DNA Clean and Concentrator kit, with elution in less than 10µL).
  • Alternatively, if the product is not clean (excessive dimer or unintended products), the PCR product can be gel purified and gel extracted (recommended kit is the Zymoclean Gel DNA Recovery Kit, elution in less than 10µL).
  • After purification of your product, obtain an accurate concentration using a method such as a Nanodrop.
  • Assemble your reaction with 50ng of backbone (largest fragment of reaction) and 100ng of your insert in a volume of 2.5µL.  Add 7.5µL of Gibson Assembly Master Mix from above and mix well.
  • Incubate your reaction at 50ºC for 1 hour.
  • Chill on ice for 1 minute.
  • Add 3uL of your Gibson Reaction to your e.coli host of choice and proceed with transformation.

Ligase Cycling Reaction (LCR)

CRISPR Editing in Cell Lines and Model Organisms

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Mammalian Cultured Cell Lines

Testing for CRISPR induced deletions in cultured cell lines may be a quick and easy way to validate your targets or designs before embarking on full mouse or other model organism deletion.  The goal of this approach may be to isolate individual clonal cell lines for further assay of your gene of interest.

If the cell line you wish to use as your model system is not able to be transfected by chemical methods or through electroporation, it may be wise to pursue the lentiviral introduction of the Cas9 and guide RNAs.  Those plasmids are listed above in the Plasmids for Consideration.

Here, we will outline an approach for targeted deletion of a 1kb segment of DNA with non-homologous end joining (NHEJ) using standard transfection techniques using in a cell line that has a transfection efficiency > 30%.  To accomplish this, we will use 2 guide RNAs (guide 1 – G1; guide 2 – G2) that flank the 1kb region we wish to delete, however, this method can be used to delete any size or piece of DNA.

  • Design and clone guide RNA sequences into a plasmids such as pX330, pX458(GFP), or pX459(puro) (guidelines here).  A good sequencing primer for pX330, pX48 derived plasmids is “CBh_pro_rev 5′-CGTCAATGGAAAGTCCCTATTGGCGT” which is a reverse primer in the CBh promoter that drives Cas9 expression and is 250bp downstream of the guide RNA cloning site OR “hU6-seq-F 5′-GAGGGCCTATTTCCCATGATTCC” which is upstream of cloning site in the human U6 promoter.
  • Co-transfect the G1 and G2 verified plasmids into your cell line of interest.  If you are using pX330 (without GFP marker), also co-transfect a plasmid that expresses high levels of GFP such as pmax-GFP (Amaxa) or other.
  • Post transfection, monitor the color in your transfected cells and the confluency.  When the cells approach a reasonable density or confluency, sort them through fluorescent assisted cell sorting (FACS) at the UW Carbone Cancer Center Flow Cytometry Laboratory.  The facility manager is Dagna Sheerar and can help assist you in appointment set up and cell sorting procedures.  Please book well in advance.  Sorting the cells will lessen the size of the haystack in which your needle (single cell) is sitting.
  • Sort from the top 3 to 10% GFP positive cells into both 96 well plates (1 cell per well) as well as collect a “bulk” sample for PCR testing/validation of targets.  The highest scoring GFP cells should be avoided as they often do not grow well or retain normal cell morphology.  The sorter will have about a 50-70% success rate in placing single cells into wells.  Score the wells after a 3-4 days for 0, 1, or 2+ cells.
  • Grow out single cell isolates in 96 wells until confluent.  Cherry pick those wells into a 24 well plates and save the rest into a microfuge tube for DNA isolation.  [20uL of trypsin per 96 well, add back 200uL complete media, pipette well, move one drop of cells into a new 24 well plate, the rest into a microfuge tube]  Test/analyze for deletions in the isolated cells before the 10% wells reach confluency.
  • Isolate the genomic DNA from each tube via protocol listed here and test by PCR with primers that span your entire deletion, span each junction of your deletion, as well as a primer set that is internal (should be completely deleted if the 1kb fragment has been deleted).  It is important to test all of these combinations as NHEJ may result in odd recombination events, including inversion of the 1kb segment.
  • Sequence those positive PCR products for the identity of your deletion.  Maintain those cells that contain your desired and complete deletion.

This method will likely not result in stable expression of your Cas9+guide RNA constructs unless you apply selective pressure with the pX459-puromycin plasmid.


If you have completed initial testing on your CRISPR targets, or you are a very brave scientist, you are now ready to make a knockout or transgenic mouse.  There are several ways that this can be completed and we will discuss a few below with which we’ve had success.

Here, we will outline an approach for targeted deletion of a 1kb segment of DNA with non-homologous end joining (NHEJ) using standard transfection techniques using in a cell line that has a transfection efficiency > 30%. To accomplish this, we will use 2 guide RNAs (guide 1 – G1; guide 2 – G2) that flank the 1kb region we wish to delete, however, this method can be used to delete any size or piece of DNA.

You may also wish to add a donor piece of DNA for homology directed recombination (HDR).  That will be discussed below.

Method 1: RNA guides + Cas9 protein pronuclear injection

  • For CRISPR mouse generation, RNA needs to be in vitro translated first from a PCR DNA template.
  • T7-additionforguide: 5’-TTAATACGACTCACTATAGG-guidesequencehere
  • Use the MEGAshortscript Kit from Ambion (AM1354) – for the Guides
  • Steps for generation of T7 RNA:
    1. PCR amplify, gel purify DNA template with primers to add T7 promoter sequence
    2. Use MEGAshortscript to generate RNA
    3. Purify RNA using the MEGAclear kit (AM1908) –
    4. Spec with Nanodrop and run on the Agilent Bioanalyzer, final spec with QuBit at GEC in Biotech Center
  • PCR to create the template for IVT
    • can use any high fidelity polymerase for reaction
    • use the T7 primers listed above (the forward primer will contain your guide sequence)
    • DpnI digest the plasmid for 1 hour
    • Gel purify the guide templates (2% agarose gel) to separate the 100bp size from any dimer.  We recommend the use of Zymo Gel Purification kit (or Qiagen Min-elute) and elution in 12uL of water.
    • Spec samples on nano-drop or similar for accurate concentration.
  • MEGAshortscript kit
    • Thaw the T7 10X Reaction Buffer, four ribonucleotide solutions, and Water at room temperature. Briefly vortex the T7 10X Reaction Buffer and ribonucleotide solutions. Microfuge all reagents briefly before opening to prevent loss and/or contamination of material that may be present around the rim of the tube. Keep the T7 Enzyme Mix on ice during assembly of the reaction.
    • Use all 8uL of PCR template if the concentration of your PCR template is below 40ng/uL, otherwise, use 300ng/uL of template
    • Assemble the reaction at room temp according to protocol included with kit
    • Mix reaction gently, quick spin to collect sample
    • Incubate at 37C for 3 hours (can go to 4 hours if necessary)
    • Add 1uL of Turbo DNaseI and incubate for 15 minutes at 37C
    • Clean up using the MEGAClear kit
  • MEGAClear kit
    • Before using the kit for the first time, add 20mL of 100% ethanol to Wash Solution Concentrate
    • Bring the RNA sample to 100uL using the Elution Solution. The samples from the MEGA Shortscript kit will need to have 80uL added.
    • Add 350uL of Binding Solution Concentrate to the sample, mix by pipetting
    • Add 250uL of 100% ethanol to the sample, mix by pipetting
    • Apply the sample to the filter -> put a filter into a collection tube, put the RNA into the filter cartridge, spin for 30 seconds at RCF 10-15k (not any harder than that), discard flow through and wash sample
    • Wash 2 x 500uL Wash Solution
    • Elute RNA from filter with 50uL of elution solution.
      1. pre-heat 110uL of Elution Solution to 95C. Apply 50uL of solution to cartridge and spin at 10-15k RCF for 1 minute
      2. Repeated a second time with 50 uL more, then collect into the same tube.
      3. RNA precipitation: Add 1:10 volume of 5M Ammonium Acetate (so 10uL if sample was 100uL), then add 2.5 volumes (275uL) of 100% ethanol, mix well and store at -20C for 30 minutes. Spin down at max speed 4C centrifuge for 15 minutes, discard supernatant, wash pellet with 500uL 70% cold ethanol (-20C), spin, remove ethanol, spin, dry pellet. Resuspend in suitable volume, most likely around 20uL, in injection buffer from Kathy.
  • Spec and assay the eluted RNA
    • Spec the RNA on the Nanodrop, should be in the range of 300-600ng/uL
    • Run Agilent Bioanalyzer to make sure you have a major peak near the 100nt region
    • Finally, run the QuBit for better RNA quantitation.  The QuBit is in the Gene Expression Center in the Biotech Center.  Make a 1:10, 1:100 and 1:500 dilution of each guides to make sure you are within the detection limits of the QuBit and this also makes the back calculations easy.
  • Injection solution:
    • The final concentration of guides should be 50ng/uL in a 50uL aliquot.  The Transgenic Facility has Cas9 protein from PNA Bio (40ng/uL final), therefore you need the guides at 50ng/uL in 48.4uL of injection buffer and the Cas9 (1.6uL) will be added to that for a full 50uL.

Method 2: pX330 (guide+Cas9) pronuclear injection


Coming Soon!