Protocols

CRISPR targeting consists of a Cas protein (usually spCas9) which creates a double strand break at a defined location and a guide RNA (gRNA) which complexes with the Cas protein to target the double strand break. If the goal of the mutation is to introduce a defined sequence such as a base change, tag or transgene, a DNA vector containing the desired mutation along with arms of homology flanking the mutation is also required. The DNA is often referred to as the homology directed repair (HDR) template 

Cas protein: The Core supplies spCas9 which is the Cas used for the vast majority of work. There are some situations where an alternate Cas is needed. In these cases, please contact Chip to discuss your experiment.

Guide RNA: The gRNA is a short RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined 20 nucleotide spacer that directs the double strand break to the desired target. The 20mer must be adjacent to a Cas defined PAM site which for Cas9 is NGG. The double strand break is made between the 3^rd and 4^th nucleotide of the spacer from the PAM. The goal for choosing a gRNA is to have the break be within 10 nucleotides of the desired mutation site. You also need to identify gRNAs that have a low likelihood of creating breaks in undesired locations. There are numerous web based applications to assist with finding gRNA 20mers. Most give the same results but present the options in different ways. One frequently used by the Core is CRISPOR. If you are not able to identify a suitable gRNA that meets your goals, please contact Chip to explore other options or to request assistance in your design.

HDR Template: The format of the HDR Template is chosen based on the desired mutation. It can be single or double stranded and consists of the desired mutation which is flanked by arms of homology. Usually, smaller is better when injecting DNA into embryos so a goal is to use the smallest DNA possible to achieve your goal. The goal also drives the format of the DNA to use…

Small Knockins or nucleotide mutations: The best HDR template format for insertions or nucleotide changes under ~130 bases is a small single strand DNA oligo. The design includes the desired mutation flanked by 35-50 nucleotide arms of homology. It’s important to disrupt CRISPR targeting along with introducing your desired mutation by mutating one of the Gs in the PAM or by making 2 or more base changes in the 20mer. If your mutation doesn’t create these conditions, use codon swaps to achieve this. The oligos are most often purchased from a vendor such as IDT. Purchase the highest purity available from the vendor to avoid contaminants that can kill the embryos. Purchase 2-4 nmol quantity and do not resuspend prior to submitting.

Large Knockin Template for Microinjection: While the HDR template for large knockins can be double or single strand DNA, single strand DNA has been found to work better. The design includes your transgene flanked by arms of homology. There is still some debate on the best size of arms – short 36 base have been shown to work well for knockins in the 1000 nucleotide range however for larger transgenes arms that are 100-500 nucleotide are probably a better choice. It’s important to disrupt CRISPR targeting along with introducing your desired mutation by mutating one of the Gs in the PAM or by making 2 or more base changes in the 20mer. If your mutation doesn’t create these conditions or insert inside the 20mer+PAM, use codon swaps to achieve this. There is flexibility on the source of the template. When purchasing or preparing the template, always keep in mind that the reagents will be injected into embryos. Impurities will prevent clog the injection pipet or kill the embryos in utero. If preparing your own template, follow the guidelines below. If purchasing from a vendor, pay attention to how the DNA is purified. For example, IDT gBlocks are a poor choice for embryo injection whereas IDT Megamers usually work well. When choosing a vendor, please discus the goal of embryo microinjection with them. The desired quantity for submission is 3ug. If purchased from a vendor, do not resuspend the prior to submitting. If preparing yourself, be sure to use injection buffer provided by the Core and the concentration should be ~100ng/ul.

Plasmids should be purified using an endoplasmid free kit such as this.

It’s important to excise your transgene from the plasmid reducing plasmid DNA as much as possible. It’s also important to submit DNA that is free from impurities that could clog the injection pipets or kill the injected embryos. The following 2 kits are popular methods for preparing DNA for pronuclear microinjection:

QIAquick Gel Extraction Kit (70 bp – 10 kb)

QIAEX II Gel Extraction Kit (40 bp – 50 kb)

With both kits, use T-lowE (10 mM TRIS, 0.1 mM EDTA) which is available from the Transgenic Core for the final elution step.

Even with kits, we see variation in the purity of the DNA submitted. The purity of the DNA and the accuracy of the quantification are the 2 most important factors in the success of your injections. Even when using a kit, careful attention to details is essential! Follow all extra steps for increased purity. The goal is to obtain 4ug DNA at 100ng/ul.

This protocol has been taken from “Manipulating the Mouse Embryo”. Hogan et al. Cold Spring Harbor Press. 1994. Although the procedure is laborious, it results in DNA samples that are easily microinjected and should yield uniformly high frequencies of embryo survival and transgenesis. To avoid accumulation of dust during the concentration of DNA, all solutions should be filtered through a 0.2 m filter and all tubes and pipettes should be rinsed with filtered water prior to use. Do not use pipette tubes packed by hand in the lab, obtain commercial tips for this procedure.

1. Isolate 50g or more of the DNA fragment to be microinjected. Excise the band from the agarose gel in a minimum volume of agarose and electroelute the DNA from the gel slice in a dialysis bag for 2-3 hours. Carefully remove the buffer and agarose from the bag (most of the DNA will adhere to the bag), add fresh buffer and reverse the polarity of electroelution for a few minutes to recover the DNA.

2. Extract DNA several times with phenol and then extract with ether three times to remove residual phenol.

3. Precipitate with ethanol several times and resuspend in a minimum volume (e.g. 50-100l) of injection buffer (T low E – 10mM TRIS, pH 7.5, 0.1mM EDTA – available from the Transgenic Core Lab). Determine DNA concentration.

4. Dissolve at least 10g of DNA in 2.4 ml of 10mM TRIS-HCl (pH 8.0), 1mM EDTA. Add exactly 3.0g ultrapure CsCl and dissolve. Check that density is 1.700.01g/ml. Adjust if necessary.

5. Transfer the DNA solution to a 1.3cm polyallomer ultracentrifuge tube, cover with light paraffin oil, and centrifuge in a SW50.1 rotor at 200C for 48 hours at 40,000 rpm.

6. Collect 0.2ml fractions from the bottom of the tube. Assay ~8 fractions from the middle of the tube by running 1-3l on an agarose minigel and pool the fractions containing DNA.

7. Dialyze at 40C against a large volume of injection buffer, changing the injection buffer several times over a 48 hour period.

8. Filter through a 0.2m filter; determine DNA concentration; dilute to 100ng/l in embryo tested injection buffer. Run a gel to confirm purity and concentration.

Our lab has injected a large number of constructs from a variety of labs and with several different ES cell lines.  We have noticed that the two most important factors that affect chimerism rates are morphology and confluency.  The colonies should be small, tight, have smooth raised edges, and in log phase growth.  Confluence should be ~60-70%, with many small to medium size colonies. The ES cells are considered overgrown when they are no longer individual colonies and have begun to run into each other.  It is important to have the cells in log phase growth because we are looking for the ES cells to overtake the embryo.  When the ES cells are overgrown and have plateaued the ES cells do not contribute as large a percentage to the embryo.  The result is lower numbers of chimeric animals and lower percentage of chimerism in the individual founders with reduced germline transmission.

Because different cell lines and clones grow at different rates it is best to prepare a least two dilutions of each clone for injection.  A simple method is to thaw your vial of cells for injection, or passage the clone, 1/3 in one well and 2/3 in the second well. If you are low in vial numbers for each clone we will be happy to return any unused wells back to you, otherwise we discard any cells that we do not use.

When you are ready for injection we would prefer at least two dilutions of each clone, with each dilution growing in one well of a 6-well plate.  To change the smallest number of variables we ask for an aliquot of your ES cell media (NO DRUGS) and trypsin.  We use that media to inject the ES cells into the embryos.  Feed the ES cells 2-3 hours before injection (8-10 AM) to make sure they are in log phase growth for injection and bring them to out lab by 11:00.  We inject the ES cells  ~ 12 AM in the morning Tuesday – Friday.  We inject up to 4 clones per paid construct.

1. Prepare lysis buffer by diluting Proteinase K stock to 250 ug/ml (cells) or 500 ug/um (tails) in appropriate quantity of buffer (500ul per sample)
2. Add 500-ul buffer to each tail sample or well of cells (this is for a 6-well plate, adjust to smaller or larger volume for other plate sizes).
3.Incubate tail samples overnight at 37ƒ C or cell samples 2 hours to overnight at 37 C.  Transfer cell samples to eppendorf tubes after incubation.
4.Add 250 ul saturated (6M) NaCl to each tube.
5.Vortex 2-5 min.
6.Place on ice for 10 min.
7.Spin 10 min. at low speed (9500 rpms on Eppendorf microcentrifuge)
8.Carefully remove top 500 ul to fresh tube with 1 ml of 100% EtOH and mix by inversion.
9.Centrifuge at high speed for 10 min. (14,000 rpm on Eppendorf microcentrifuge)
10.Remove supernatant by vacuum and wash with 70% EtOH 2X to remove salts (200 ul/wash).  Dry.
11.Resuspend in 50-100 ul of TE or H2O O/N at room temp.
Reagent
per 50 mls:
Final Concentration
1 M Tris, pH 8
500 ul
10 mM
5 M NaCl
1 ml 
100 mM
0.5 M EDTA, pH 8 
1 ml 
10 mM
10% SDS
2.5 ml
0.5 %
H2O
to 50 mls

This protocol is for high quality DNA for Southern analysis for screening potential hetero- or homozygotes.

   1. Cut 1 cm of mouse tail and place in 1.5 ml eppendorf tube
   2. Add 500 ul of 50 mM Tris (pH 8), 100 mM EDTA, and 0.5% SDS
   3. Add 25 ul of 10 mg/ml of proteinase K and mix well
   4. Incubate at 55 C in a waterbath overnight
   5. Next day add 500 ul of phenol and rotate for 20 min. at room temp.
   6. Centrifuge at 14,000 rpm for 3 min at room temp
   7. Take off supernatant and repeat steps 5-6
   8. Take supernatant and add 500 ul of phenol/chloroform (1:1) and rotate for 20 min
   9. Spin at 14,000 for 5 min
  10. Transfer the top aqueous layer to a clean tube.
  11. Add 50 ul of 3 M sodium acetate (pH 6) and 500 ul of 100 % ethanol. Mix well, (you should be able to see DNA precipitate)
  12. Remove as much of ethanol supernatant as possible
  13. Wash pellet with 1 ml of 70% ethanol 2X
  14. Spin in microcentrifuge at 14,000 for 1 min
  15. Aspirate supernatant carefully using a pipette tip, dry with speed vac if available
  16. Add 100 ul of water and store at 4ƒ C overnight to dissolve.

The purpose of the School of Medicine Transgenic Mouse Core Laboratory (TCL) is to make genetically modified mice for Johns Hopkins University researchers.

1. Eligibility. Approval of the Institutional Animal Care and Use Committee (IACUC) is needed to breed, house and perform experiments with mice at JHU.  Forms for protocol submission to the IACUC are available from Comparative Medicine (955-3273).  Do not include a description of the procedures involved in the production of transgenic mice since that is covered by the TCL protocol.  A current protocol number, representing IACUC approval is required to initiate a procedure.

2. Priority. DNA constructs will be injected for JHU investigators on a first come, first served basis with the caveat that an investigator may only have 2 constructs in the queue at any time.  If the investigator submits more than 2, the additional constructs will be held off the queue until one of the first 2 is started.  Only then will an additional construct be added to the queue.

3. Responsibilities of the TCL:  The Transgenic Core will inject enough embryos to implant ~25-30 embryos into each of 10 recipient females. This usually requires the injection of 400-600 embryos over 2-4 days.  This level of injection will usually result in 20-40 pups being born. Approximately 10% of pups born tend to be transgenic. The birth and transgenic rates are very dependent on the quality of the DNA preparation and the accuracy of the concentration determination. If a greater than average amount of embryo death is occurring, we will ask the researcher to re-prepare the DNA. Poorly prepared DNA is thought to be the most common reason for poor numbers of pups being born in a well established microinjection lab.

4. Investigators are responsible for identifying transgenic pups and notifying the TCL of the results. The TCL will attempt to transfer the Moms and pups to the investigator when the pups are 1-2 weeks of age. It will be the responsibility of the investigator’s laboratory to wean (if not already done) and screen the pups for the presence of the transgene. If a round of injections fails to produce at least 2 founders, the TCL will re-inject the same construct one time. In order to be eligible for re-injection, the animals must be screened and a re-injection request made within 6 weeks of the transfer of the pups to the investigatorís animal colony. In order to re-inject, the person making the request must be able to demonstrate that the screening protocol is able to identify transgenic founders. Positive controls should include multiple dilutions of plasmid into a wild type mouse DNA prep (at the same concentration of total DNA as the potential founder preps), down to a plasmid concentration of 10fg/ul.

5. Maintaining virus antibody-free mice. The TCL and Animal Services are following procedures to maintain disease-free animals.  Our lab follows an extensive sentinel screening program in which every rack is screened monthly.  Additionally, every litter of pups is tested for MHV, Helicobacter sp. and pinworms prior to being transferred.  Currently our mice can be shipped to any animal facility at Hopkins directly except for CRB.  Mice going to CRB must first be shipped to BRB for additional monitoring.