Transgenics & Knockouts CRISPR

The majority of genetically modified mouse lines are now generated in our unit using CRISPR/Cas9 technology. Although since 2014, we have been routinely producing CRISPR genetically modified mice with a high degree of success, the technology is evolving at a rapid pace and we continue to evaluate modifications of methods that may significantly improve efficiency.

Below is information which will be helpful in beginning your in vivo CRISPR project.

Planning Your Project

I would recommend that you plan your guides and repair templates, and genotyping strategy, together with our experts in CRISPR planning from Bioinformatics dept.  Contact Shifra Ben Dor ext. 2470.

Useful sites

• LSCF Bioinformatics - Target/DNA
• Addgene: CRISPR Guide
• http://www.genome-engineering.org/crispr/?page_id=27
• Genome Engineering using CRISPR/Cas Systems - ‏ Google Groups
• The CRISPR web page
• Genome Engineering Using the CRISPR-Cas9 System

Guide RNAs

synthetic crRNA and tracrRNA molecules

Once you have designed your guide sequences, we recommend purchasing synthetic crRNA and tracrRNA molecules.
See
Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins
Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes
Gene Editing in Mouse Zygotes Using the CRISPR/Cas9 System

Synthetic guides are inexpensive, and remove the necessity to produce your own guides by in vitro transcription.  
They may be purchased from IDT and other companies.
Order
Alt-R® CRISPR-Cas9 crRNA, 10 nmol
Alt-R® CRISPR-Cas9 tracrRNA, 5 nmol

HDR Oligos

We recommend oligos from IDT, 4nmole ultramer, normal purification and they generally work well. It is essential to mutate the PAM site in the repair oligo, to prevent the oligo being targeted by Cas9. If the guide is inside an exon, silent mutations can be made to prevent change in the amino acid sequence.
IDT have found that efficiency is increased if:

• the mutation is 5’ of the PAM site
• the centre of the HDR oligo is the cut site
• the mutation is 1-10 bases from the cut site. 10-20 bases gives OK efficiency, >20 bases very inefficient.

Cas9 Nuclease

We use Cas9 nuclease which we purchase from IDT
Alt-R® S.p. Cas9 Nuclease 3NLS

Genotyping Strategies

These must be built in to your original design. Whilst an RFLP to identify editing is attractive for an initial screen, it is our experience that it is not unusual to fail to identify the RFLP even when editing has occurred. For HDR we suggest sequencing all the target sites to identify HDR events or INDELs, which will indicate whether the guides are working even if there has been  no HDR repair. Primers for PCR for sequencing should be tested in advance on wild-type mice, so that a calibrated screen is in place before embryos/tails are received for genotyping. 

Off-targets

CRISPR mice have been reported to have very much fewer off-targets then cell lines:

• Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC. Nat Methods. 2014 Apr;11(4):399-402.
• Off-target mutations are rare in Cas9-modified mice. Iyer V, Shen B, Zhang W, Hodgkins A, Keane T, Huang X, Skarnes WC. Nat Methods. 2015 May 28;12(6):479

NOTE: there have been projects we have done, where sequencing has revealed at least one of the potential off-targets was mutated in the CRISPR founder mouse. Note that as long as the off-target is not linked on the same chromosome to the mutation of interest, off-targets can be removed by outcrossing the mice.

A recommendation is to generate more than one line of mice carrying the same mutation, but using different guide RNAs for targeting. This will ensure that the observed phenotype is caused by the mutation in the gene of interest and not by off-target mutations.

Conditional Alleles

Insertion of LoxP sites to flank an exon (exons) of interest is possible using CRISPR technology, but can be problematic. The most common method involves delivery to the embryo of two separate sgRNAs, targeted to sequences flanking an exon of interest and two ssODN donors, each containing a LoxP site flanked by short (40–80 bases) arms homologous to the desired insertion site. Both Loxp sites must be inserted into the same allele. Efficiency tends to be low, and a variety of mutations, including single LoxP insertions, double LoxP insertions located in trans, deletions resulting from non-homologous end-joining (NHEJ) and insertion of mutated LoxP sites are commonly detected.
Alternative strategies involving plasmid or long single-stranded DNA molecules can be discussed.

Mouse Strains for Injection

Our standard default strain is C57BL/6, we use C57BL/6JOlaHsd from Envigo.
The inbred strain FVB also works well.
Other backgrounds can be discussed if required.

IACUC and Internal Service Orders

Clients not from the Weizmann Institute should contact Rebecca Haffner.

NOTE: We will not schedule an injection session until all reagents have been delivered to me and an order has been made in Internal Services.

(Test Injections)
Before we do the full injection, we can inject a number of embryos (about 50), and leave them to develop in vitro to blastocyst stage. They can then be analysed for the presence of the mutation, and for indels at the cut sites.

PRACTICAL NOTES

Reconstituting  synthetic RNAs (crRNA and tracrRNA).
For each of the reagents you receive, (both crRNA and tracrRNA), resuspend each reagent in IDT’s Duplex Buffer at a concentration of 100 uM. Use IDT’s resuspension calculator for volumes.
Be scrupulous about RNase free conditions (gloves, new box tips, tubes etc).

Anneal crRNA and tracrRNA
Prepare guide RNA complex by mixing equal volumes of 100um crRNA and 100um tracrRNA and anneal in a thermocycler (95^oC for 5 min and then ramp down to 25^oC at 50^oC/min). Alternatively, incubate the tube 95^oC and let it cool to room temperature on the bench. Aliquot immediately into 3ul aliquots in 1.8ml Eppendorf tubes. Label guide clearly 50 uM cr+tracr + name of guide.

ss HDR donor oilgos
Resuspend donor oligos in RNase free water at a concentration of 100 uM. Aliquot into 10 ul aliquots in 1.8ml Eppendorf tubes. Label guide clearly 100 uM ssODN + name of donor.

We require 6 aliquots of annealed cr+tracr RNA and 2 aliquots of HDR oligo.

Please coordinate with me, to obtain a form which must be submitted with the CRISPR reagents. RNAs must be brought over on dry ice, together with a Revco box for storage and the printed filled form. Write the name of the experiment and the lab on the SIDES of the box!

Remember: Be scrupulous about RNase free conditions (gloves, new box tips, tubes etc) at all stages.