CRISPRs through the GRCF
CRISPRs have transformed the world of genome editing, opening up great potential and exciting opportunities for understanding the relationships between genes and phenotypes. At the GRCF, we have partnered with three innovative companies to provide you a broad range of products to assist you in every aspect of this rapidly moving field. Agilent provides custom sgRNAs for CRISPR with full in-house support. Horizon/Dharmacon and IDT both provide online tools for design assistance.
Horizon/Dharmacon and IDT products may be ordered directly from their portals. Agilent products require a custom quote. Please fill out the linked order form and email it to the GRCF. We will connect you with Agilent.
Advantages to CRISPR over ZNF or TALEN:
The sequence of the guide RNA that is a part of the Cas9 protein can be easily and relatively cheaply designed.Efficacy is high and much faster with CRISPR due to the Cas9protein being able to be directly injected into developing embryos rather than traditional homologous recombination techniques.
Due to the above advantages, CRISPR can effectively introduce mutations in multiple genes in one step, which allows for rapid turnaround in genome engineering. Changes that previously were introduced one at a time over multiple generations can now be introduced simultaneously in one generation, greatly increasing the ability to study the effects of the changes and develop hypotheses.
Introduction to CRISPR:
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a method of gene editing that utilizes the Cas9 protein and specific guide RNAs to either disrupt host genes or insert sequences of interest. Initially used in bacteria as an adaptive immunity response, CRISPR has been since utilized in the biological field as a new alternative to genome engineering. Furthermore, CRISPR provides a cheaper alternative to other gene editing techniques such as zinc fingers, and is quickly being adopted as the technique of choice.
Cas9 (CRISPR associated protein 9) is a naturally occurring enzyme found in some bacteria that is used for immunity. Cas9 works by using guide RNA with short sequences complimentary to potential foreign DNA, combating infection. This mechanism has similarities to RNA interference found in many eukaryotes. Because of its capabilities, Cas9 has been used recently in experiments to serve as a genome editing tool.
How CRISPR works:
Cas9 with designed guide RNA[s] are injected into developing embryos.Once inside the cell body, Cas9 searches through the cell’s for a DNA sequence that corresponds to the sequence in the designed guide RNA.Once the sequence is found, Cas 9 is able to unwind the DNA helix.After DNA has been unwound, Cas9 creates a double stranded break in the cell’s DNA.Repair enzymes repair the double-stranded break while integrating new experimental DNA.
Frequently asked questions
Ordering lentivirus vectors from this site requires that investigators have a current recombinant DNA and infectious agent registration for this material from the Johns Hopkins Biosafety Office. The other components do not have any special requirements.
Since CRISPRs have been shown to have a wide variation in their activity, even when designed in close proximity to each other, it is recommended you order at least 2 gRNAs per target.
Standard oligo orders placed before noon from MilliporeSigma arrive the next business day. Standard oligo orders placed with IDT before 2 PM will arrive in our office two business days after they are placed. Other items vary depending on the time it takes the manufacturer to ship. All items are available in Blalock 1004 the day they are received and will go out for delivery with the CoreStore the next morning.
No. Due to the sensitive nature of some of the items ordered through our portals, someone must be present in your lab for your order to be delivered.
Yes. On a typical day, we receive the orders by 11:30 AM. Orders are available in Blalock 1004 from the time they arrive until we close at 4:30 PM. You may stop by and pick them up anytime during those orders. If you call ahead, please be prepared to tell us the order number and then name of the person placing the order. Orders are brought to the CoreStore early the following morning and set out for delivery.
If you know that you would like to pick up your order at the time you place your order, you may replace your building and room number with the words “Pick up” and we will contact you when the order arrives. Pickup orders are placed in one of our lockers near the Blalock elevators and are available 24/7.
- For Dharmacon, if you have a current account, you do not need to create a new account. Simply starting on our portal login page will associate your account with our portal. If you need to create an account, you can do so on the registration page. Orders created on the Dharmacon portal create a quote which is emailed to the GRCF. The GRCF will place the orders by the end of the day. You will receive an email when the order has been placed.
- For IDT, you must create a portal specific account, but account set-up is instantaneous. Simply go to our portal and create a new account. Make sure you do not click anywhere else on the page prior to creating the account, or the web-site will lose the association with our portal. Once you have set up the account, you may place your order. When placing your first order on a newly created account, you will see incorrect pricing and shipping charges, but your bill will be correct.
- For MilliporeSigma, you need to email us the following information:
- Your name
- Your email address
- Your phone number
- Your building and room number (for CoreStore deliveries)
- Your PI’s name & email address
- If you already have set up a GRCF account with MilliporeSigma, you may order using this link.
- ThermoFisher and Qiagen do not require an account. Shopping carts are emailed to the GRCF (email@example.com) for ordering.
Dharmacon and IDT are instantaneous. If you already have a Dharmacon direct account, you may simply login on our portal page and your orders will be associated with the portal. If you already have a direct IDT account, you MUST create a new account by clicking “Set up new account” on our portal page (without clicking anywhere else on the page.)
For MilliporeSigma, you must create an account and wait for an email indicating that the account is active. This may take up to two business days.
ThermoFisher and Qiagen do not require an account. Orders are emailed to us and placed by the GRCF staff.
No. You must create an account specific to the GRCF portal in order to take advantage of our pricing. The exception to this is Dharmacon. Prior Dharmacon accounts work, as long as you start on our portal page.
On any of these sites, you will pay with your budget number. You do not need a credit card or a PO number from SAP. KKI customers should enter the last name of the PI on their grant or a KKI PO number. All customers (KKI and JHU) will be billed through iLab. JHU customers should check to make sure their budget numbers are active in iLab. Non-sponsored budget numbers must be added to iLab manually by following these instructions.
You should create a separate ordering account for each lab to make sure that your orders are delivered to the correct location.
This usually means that you have logged into the portal using a username and password that were set up for the SAP punchout or the normal IDT web-site. Using the wrong login will take you to the direct website without any notification. You will only receive free shipping if you use a username and password set up specifically on our portal. You will know you are on the correct website if you see a banner that says “You are using the DNA Analysis Facility Portal” on every page. Also, when you begin the checkout process, the billing and shipping information should be prepopulated with the GRCF address and PO. Click past this page and you will come to a page that asks for your budget number and building and room number.
Johns Hopkins East Baltimore Medical Campus
600 N. Wolfe Street
Blalock Building, Room 1004
Baltimore, MD 21287 USA
Monday – Friday:
7:00 am to 4:30 pm
After-hours pickup is available from the lockers near the Blalock elevators. Please contact us to make arrangements.