1.3.1 Make chemo-competent bacteria: Streak out DH10B bacteria on an LB-agar plate without antibiotic, incubate at 37C over night
Inoculate a liquid culture in LB medium from a single colony. Shake at 37C over night
Inoculate 250ml of LB +20mM MgSO4 with 2.5ml of the overnight culture. Grow until OD reaches 0.4-0.6
Pellet bacteria at 4C and resuspend in 100ml buffer A (30mM potassium acetate, 10mM CaCl2, 50mM MnCl2,
100mM RbCl, 15% glycerol, pH 5.8 adjusted by acetic acid) on ice
Incubate the cells on ice for 5 minutes, pellet at 4C, resuspend in 10ml buffer B (100mM MOPS, 75mM CaCl2, 10mM RbCl,
15% glycerol, pH 6.5 adjusted with KOH) on ice
Incubate cells on ice for 30 minutes, aliquot into 50�l per tube, snap-freeze in liquid nitrogen
Store at -80C
1.3.2 Digest backbone plasmid: 5�l FastDigest Green buffer
8�g plasmid DNA (pCAS9-mCherry empty)
to 46�l water
2�l FastDigest ApaI
2�l FastDigest BcuI
Incubate for 3h at 37C in a thermocycler
Column-purify using AnalytikJena PCR purification kit or similar; elute in 15�l water
1.3.3 Chew-back 3' ends: 10�l 10x NEBuffer 2
10�l column eluate
1�l dTTP 100mM
1�l BSA 10mg/ml
3.33�l T4 DNA polymerase (3 U/�l)
Incubate for 5 minutes at 27C in a thermocycler, put on ice
Incubate for 20 minutes at 75C in a pre-heated thermocycler
1.3.4 Prepare cloning mastermix: add 5�l Constant gRNA antisense oligo (100�M)
add 10�l 10x NEBuffer 2
Store cloning mastermix at 4C for up to 3 months
1.3.5 Assemble gRNA construct: Dilute gene-specific gRNA oligo (100�M) 1:100 in water
Mix 2�l of oligo dilution with 2�l of cloning mastermix
In parallel, prepare a water control (2�l water + 2�l mastermix)
Anneal in a thermocycler using the following temperature ramp:
75C -> 55C at 2.5 minutes per degree Celsius
55C for 30 minutes
55C -> 25C at 2.5 minutes per degree Celsius
1.3.6 Transform bacteria: Thaw 2x 50�l competent bacteria on ice
Add 4�l assembly per tube, flick the tube
Heat to 42C for 1 minute
Cool on ice for 2 minutes
Streak on LB-Amp Agar plates, incubate at 37C over night
The next day, the water control should contain >20x less colonies than the real assembly
3.7 Expand a clone: Inoculate a LB-Amp liquid culture and shake it over night at 37C
Perform a miniprep and sequence the plasmid DNA using the primer U6-seq
The sequencing result has to contain the complete gene-specific gRNA oligo sequence
1.4.2 Single cell cloning: Remove the medium, add 30�l 0.05% Trypsin solution to the well, incubate for 5 minutes at 37C
Add 200�l medium and resuspend
Count a fraction of the cells
Plate cells at a density of 0.5 cells per well into 10 96-well plates
Grow clones for 1-3 weeks until they on average cover a quarter of the well
1.4.3 Clone picking and duplication: Trypsinize 96 wells with a growing clone in them
Resuspend clones in 200�l medium and transfer to a new 96-well plate
When the plate is full, transfer 100�l from each well to a second 96-well plate
Incubate cells for 2 days at 37C
1.5.1 Lysis of the cells: Prepare a stock of a 1x direct lysis buffer (1 mM CaCl2, 3 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10 mM Tris pH 7.5)
Add 0.2 mg/ml Proteinase K to an aliquot of the lysis buffer
Remove the medium from one copy of the cell clones
Add 30�l lysis buffer and resuspend, transfer to a 96-well PCR plate
Incubate for 10min at 65C and for 15min at 95C, then store on ice
1.5.3 Preparation of barcode primer plate: Pipet 2.5�l of each of the primers D501-D508 into all wells of rows A-H, respectively, of a round-well 96-well plate
Pipet 2.5�l of each of the primers D701-D712 into all wells of column 1-12, respectively, of the plate
Add 95�l water to each well of the plate and resuspend
1.5.4 Second PCR Prepare a 120x PCR mastermix containing:
- 300�l Phusion HF buffer
- 30�l dNTP mix 10mM
- 15�l Phusion polymerase
- 615�l water
In a 96-well PCR plate, mix 2�l first PCR, 2.5�l barcode primer mix, and 8�l mastermix. Avoid contaminating the barcode plate.
Run with the protocol: 2min 98C, 19x(20sec 98C, 20sec 60C, 30sec 72C), 2min 72C
1.5.5 Prepare DNA for a MiSeq run: Mix all PCR products in a single tube
Run PCR products on a 1% agarose gel, cut the 400-500bp band, purify it using a column purification kit, and elute in 200�l water
Precipitate by adding 1�l glycogen, 20�l 3M NaOAc pH 5.2, 220�l isopropanol, incubate for 20minutes on ice
Spin for 15 minutes at 4C, remove supernatant, wash with 70% ethanol, spin for 5 minutes, remove most liquid
Pulse spin, remove remaining liquid, dry pellet until it gets transparent
Resuspend in 35�l water for 5min at 37C, spin 3min full speed, carefully transfer 25�l from the top to a fresh tube
Measure the concentration twice using a NanoDrop spectrometer
1.5.6 Start a MiSeq run Thaw a MiSeq 300 cycles v2 cassette in a waterbath for 1h
Rename this samplesheet to the name of the cassette (typically MS...-300V2) and copy it to the folder D:\Illumina\...
Mix appropriate amounts of all DNA samples to be sequenced into 350�l of Illumina hyb buffer. 32ng of DNA roughly give rise to 1 mio reads. For genotyping 96 clones, anything above 200,000 reads would be sufficient.
Mix 10�l of DNA dilution with 10�l 0.2N NaOH, incubate for 5 minutes at room temperature, then add 980�l hyb buffer and store on ice
Mix 180�l of denatured DNA with 420�l of hyb buffer in a fresh tube and transfer completely to the sample well of the reagent cassette. Store cassette on ice.
Clean flow cell with water and ethanol and insert into the MiSeq. Insert cassette and buffer flask, empty the waste container and start the standard sequencing program.
1.5.7 Retrieve and analyze the data: When the run is finished after about 22h, follow the wash instructions on the MiSeq screen
Copy the 384 zipped FASTQ files from the folder D:\Illumina\Miseq output\-your-run-\data\intensities\basecalling to a USB drive
Copy the files to your computer and unzip them (double-click on a mac), then delete the zipped files
Click ANALYZE DATA below to analyze sequencing data and follow the instructions
2. Analyze Sequencing Data:
Nuclease Target Site (for double-nickases and TALENs both targetsites must be spanned):
OutKnocker 2.0 is developed by J. Schmid-Burgk in the group of Feng Zhang at Broad Institute of MIT and Harvard.
The following example FASTQ file can be downloaded by right klicking: example file. In this example, the human BIRC3 gene was targeted by CRISPR/Cas9 genome editing. The corresponding amplicon and target site sequences are entered above per default.
Institute of Molecular Medicine
University Hospital Bonn
53127 Bonn, Germany
OutKnocker was successfully tested on:
Mozilla Firefox version 47.0
Google Chrome version 35.0
Opera version 20.0
OutKnocker 1.0 can be redistributed and/or modified under the terms of the GNU General Public License (Version 2), as published by the Free Software Foundation. A copy of the license can be found online at www.gnu.org/licenses. OutKnocker is distributed in the hope that it will be useful, but without any warranty; without even the implied warranty of merchantability or fitness for a particular purpose. See the GNU General Public License for more details.
We kindly request that use of this software be cited in publications as:
Jonathan L. Schmid-Burgk, Tobias Schmidt, Moritz M. Gaidt, Karin Pelka, Eicke Latz, Thomas S. Ebert, and Veit Hornung. Genome Research 2014
Outknocker V1.31 (07/2016)
- Sequence logo is shown also for failed alignment reads
Outknocker V1.3 (06/2016)
- Navigation menu
- A mutation statistics table can be generated allowing to assess polyclonal genome editing
- Print / save as PDF
- Small pie charts are clickable
Outknocker V1.2 (06/2015)
- Memory use is fixed to roughly 200 MB, sequence alignment visualization is always enabled
- Mutation threshold for pie charts is fixed to 0.1% for better visualization of sequencing quality
Outknocker V1.1 (05/2015)
- Large files >1 GB can be analyzed
- Pie charts and alignments can be saved as SVG images
- Minor bases are included in the alignment sequence logo
- Small letters are allowed in the reference sequence and target site. Antisense target site sequences are allowed.
Outknocker V1.0 (07/2014)
- Public release of OutKnocker.org
The following data protection declaration applies to the use of our online offer OutKnocker.org (hereinafter "Website").
We attach great importance to data protection. Your personal data is collected and processed in compliance with the applicable data protection regulations, in particular the General Data Protection Regulation (GDPR). We collect and process your personal data in order to be able to offer you the above-mentioned portal. This statement describes how and for what purpose your data is collected and used and what options you have in connection with personal data.
Responsible for the collection, processing and use of your personal data within the meaning of Art. 4 No. 7 DSGVO is
53113 Bonn, Germany, firstname.lastname@example.org
If you wish to object to the collection, processing or use of your data by us in accordance with these data protection regulations as a whole or for individual measures, you can address your objection to the entity responsible.
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2 General website usage
The hosting services we use serve to provide the following services: Infrastructure and platform services, computing capacity, storage and database services, security services and technical maintenance services that we use to operate the Site.
We or our hosting provider process inventory data, content data, usage data, meta- and communication data of customers, interested parties and visitors of this online offer on the basis of our legitimate interests in an efficient and secure provision of this online offer according to Art. 6 Para. 1 S. 1 f) DSGVO in connection with Art. 28 DSBER.
2.2 Access data
We collect information about you when you use this website. We automatically collect information about your usage behavior and interaction with us and record data about your computer or mobile device. We collect, store and use data about every access to our online offer (so-called server log files). Access data includes, inter alia:
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Date and time of retrieval
transferred data volume
Message about successful retrieval (HTTP response code)
Browser type and browser version
Referer URL (i.e. the previously visited page)
Websites accessed by the user's system through our website
Internet service provider of the user
IP address and the requesting provider
We use this log data without allocation to your person or other profiling for statistical evaluations for the purpose of operation, security and optimisation of our online offer, but also for anonymous recording of the number of visitors to our website (traffic) and the extent and type of use of our website and services. Based on this information, we can analyze traffic, troubleshoot and correct errors, and improve our services.
This is also our legitimate interest pursuant to Art 6 para. 1 sentence 1 f) DSGVO.
We reserve the right to check the log data subsequently if there is a justified suspicion of illegal use based on concrete evidence. We store IP addresses in the log files for a limited period of time if this is necessary for security purposes. We will delete the IP address if it is no longer required for security purposes. We also store IP addresses if we have a concrete suspicion of a criminal offence in connection with the use of our website.
We do not use so-called session cookies or other cookies.
If you contact us (e.g. via contact form or e-mail), we store your details for processing the enquiry and in the event that follow-up questions arise.
This is also our legitimate interest pursuant to Art 6 para. 1 sentence 1 f) DSGVO.
We only store and use further personal data if you give your consent or if this is legally permissible without special consent.
2.5 Storage time
Unless specifically stated, we only store personal data for as long as is necessary to fulfil the purposes pursued.
3 Rights of the data subject
According to the applicable laws, you have various rights regarding your personal data. If you wish to assert these rights, please send your request by e-mail or by post to the address specified in section 1, clearly identifying yourself.
Below you will find an overview of your rights.
3.1 Right to confirmation and information
You have the right to receive confirmation from us at any time as to whether personal data relating to you will be processed. If this is the case, you have the right to request from us free of charge information about the personal data stored about you together with a copy of this data. Furthermore, there is a right to the following information:
the categories of personal data being processed;
the recipients or categories of recipients to whom the personal data have been or are still being disclosed, in particular recipients in third countries or international organizations;
if possible, the planned duration for which the personal data will be stored or, if this is not possible, the criteria for determining this duration;
the existence of a right to have your personal data concerning you corrected or deleted or to have the data controller restrict or object to such processing;
the existence of a right of appeal to a supervisory authority;
if the personal data is not collected from you, all available information about the origin of the data;
the existence of automated decision-making, including profiling in accordance with Article 22(1) and (4) DSGVO and - at least in these cases - meaningful information on the logic involved and the scope and intended effects of such processing for you.
If personal data are transferred to a third country or an international organization, you have the right to be informed of the appropriate guarantees in accordance with Art. 46 DSGVO in connection with the transfer.
3.2 Right to rectification
You have the right to request us to correct any inaccurate personal data concerning you without delay. Taking into account the purposes of the processing, you have the right to request the completion of incomplete personal data, also by means of a supplementary declaration.
3.3 Right to erasure ("right to be forgotten ")
Pursuant to Art. 17 para. 1 DSGVO, you have the right to demand that we delete personal data concerning you without delay, and we are obliged to delete personal data without delay if one of the following reasons applies:
personal data are no longer necessary for the purposes for which they were collected or otherwise processed.
you withdraw your consent, on which the processing was based pursuant to Art. 6 para. 1 sentence 1 a) DSGVO or Art. 9 para. 2 a) DSGVO, and there is no other legal basis for the processing.
you file an objection to the processing pursuant to Art. 21 para. 1 DSGVO and there are no overriding legitimate grounds for the processing, or you file an objection to the processing pursuant to Art. 21 para. 2 DSGVO.
The personal data have been processed unlawfully.
the deletion of personal data is necessary to fulfil a legal obligation under Union law or the law of the Member States to which we are subject.
The personal data was collected in relation to information society services offered in accordance with Art. 8 para. 1 DSGVO.
If we have made the personal data public and we are obliged to delete them pursuant to Art. 17 para. 1 DSGVO, we will take appropriate measures, including technical measures, taking into account the available technology and implementation costs, to inform those responsible for data processing who process the personal data that you have requested them to delete all links to this personal data or copies or replications of this personal data.
3.4 Right to restriction of processing
You have the right to request us to restrict processing if one of the following conditions is met:
you dispute the accuracy of your personal data for a period of time that enables us to verify the accuracy of your personal data,
the processing is unlawful and you have refused to delete the personal data and have instead requested the restriction of the use of the personal data;
we no longer need the personal data for the purposes of processing, but you do need the data to assert, exercise or defend legal claims, or
you have filed an objection against the processing pursuant to Art. 21 para. 1 DSGVO, as long as it is not yet clear whether the justified reasons of our company outweigh yours.
3.5 Right to data portability
You have the right to receive the personal data concerning you that you have provided to us in a structured, current and machine-readable format, and you have the right to transmit this data to another person in charge without our interference, provided that
processing is based on consent pursuant to Art. 6 para. 1 sentence 1 a) DSGVO or Art. 9 para. 2 a) DSGVO or on a contract pursuant to Art. 6 para. 1 sentence 1 b) DSGVO and
processing is carried out using automated methods
When exercising your right to data transferability in accordance with paragraph 1, you have the right to request that the personal data be transferred directly by us to another person responsible, insofar as this is technically feasible
3.6 Right to object
You have the right to object at any time, for reasons arising from your particular situation, to the processing of personal data concerning you on the basis of Art. 6 para. 1 sentence 1 e) or f) DSGVO; this also applies to profiling based on these provisions. We no longer process personal data unless we can prove compelling grounds for processing that outweigh your interests, rights and freedoms, or the processing serves to assert, exercise or defend legal claims.
If we process personal data for direct marketing purposes, you have the right to object at any time to the processing of personal data concerning you for the purpose of such advertising; this also applies to profiling, insofar as it is associated with such direct marketing.
You have the right to object to the processing of personal data concerning you, for scientific or historical research purposes or for statistical purposes in accordance with Art. 89 para. 1 DSGVO, for reasons arising from your particular situation, unless the processing is necessary to fulfil a task in the public interest
3.7 Automated individual decision-making, including profiling
You have the right not to be subject to a decision based exclusively on automated processing - including profiling - that has legal effect against you or significantly impairs you in a similar manner.
Automated decision-making based on the personal data collected does not take place.
3.8 Right to revoke consent under data protection law
You have the right to revoke your consent to the processing of personal data at any time.
3.9 Right of appeal to a supervisory authority
You have the right of appeal to a supervisory authority, in particular in the Member State where you are staying, working or suspected of having infringed the law, if you believe that the processing of personal data concerning you is unlawful
4 Security of your personal data
We make every effort to ensure the security of your data in accordance with the applicable data protection laws and technical possibilities.
The servers we use are regularly and carefully secured
5 Transfers of personal data to third countries or international organizations, no transfer to Non-EU-countries
In principle, we only use your personal data within our company.
In the event that we outsource certain parts of data processing ("server hosting"), we contractually oblige contractors to use personal data only in accordance with the requirements of data protection laws and to ensure the protection of the rights of the data subject.
Data transmission to places or persons outside the EU outside the cases mentioned in paragraph 2 of this declaration does not take place and is not planned.