When you've decided which gene you'd like to manipulate, your first task is to find a suitable single guide RNA (sgRNA) to target the gene. Assuming your goal is to create non-functional alleles of your chosen gene, you should select a gRNA that will target either: 1) A critical region of the gene, or 2) The 5' portion of the gene, such that mutations generate a frameshift resulting in the failure to properly translate the remainder of the gene.
The next consideration is to find sequence elements necessary for sgRNA transcription and binding. Specifically, you need to find a GGN(19)GG motif. The first GG dinucleotide is important for the T7 polymerase to efficiently transcribe the sgRNA (GA can be used for Sp6 transcription). The second GG dinucleotide is critical to satisfy the protospacer-adjacent motif (PAM) of Cas9, which requires that a NGG sequence immediately 3' of the gRNA binding site. (Note that this is true for S. pyogenes Cas9, and future versions may allow different sequences.) Ensure that there aren't sequence variants/SNPs within this site, and be sure you're designing in coding sequence not intronic sequence. Additional resources have been generated for identifying sgRNA binding sites. For example, see the Schier lab website in the Resources tab.
We've moved to a simple PCR-based method to generate sgRNAs. Check out this paper from Varshney & Burgess:
http://genome.cshlp.org/content/early/2015/06/03/gr.186379.114 doi: 10.1101/gr.186379.114
This works very well in our hands. It speeds the production of guide RNAs to a day and saves money to boot.
The next consideration is to find sequence elements necessary for sgRNA transcription and binding. Specifically, you need to find a GGN(19)GG motif. The first GG dinucleotide is important for the T7 polymerase to efficiently transcribe the sgRNA (GA can be used for Sp6 transcription). The second GG dinucleotide is critical to satisfy the protospacer-adjacent motif (PAM) of Cas9, which requires that a NGG sequence immediately 3' of the gRNA binding site. (Note that this is true for S. pyogenes Cas9, and future versions may allow different sequences.) Ensure that there aren't sequence variants/SNPs within this site, and be sure you're designing in coding sequence not intronic sequence. Additional resources have been generated for identifying sgRNA binding sites. For example, see the Schier lab website in the Resources tab.
We've moved to a simple PCR-based method to generate sgRNAs. Check out this paper from Varshney & Burgess:
http://genome.cshlp.org/content/early/2015/06/03/gr.186379.114 doi: 10.1101/gr.186379.114
This works very well in our hands. It speeds the production of guide RNAs to a day and saves money to boot.
CRISPR injections
Combine sgRNA, Cas9 mRNA, and marker:
3 µL sgRNA (~1-3 µg/µL final concentration)
1 µL Cas9 mRNA (300 ng/µL final concentration)
1 µL injection marker (e.g. 0.5% Texas Red Dextran)
Backfill ~1 µL of CRISPR mix into needles. The injection procedure goes the same as for transgenics; detailed in the "Microinjections" section. Be sure to save at least 10 eggs as uninjected to determine the overall viability of the brood.
Consider sacrificing a few injected fish during development to confirm that CRISPR results in indels in your gene of interest. By just a few days of age, there should be a sufficient number of cells to PCR from.
Allow the eggs to develop in the 6-well plates until there is just a little yolk left, and then transfer them to small tanks.
Consider sacrificing a few injected fish during development to confirm that CRISPR results in indels in your gene of interest. By just a few days of age, there should be a sufficient number of cells to PCR from.
Allow the eggs to develop in the 6-well plates until there is just a little yolk left, and then transfer them to small tanks.
Genotyping CRISPR fish
Extract genomic DNA
Take finclip from fish at ≥4 weeks of age.
Add 180 µl of NaOH (50 mM) to each tube, keep at 95º for 10-15 minutes.
Heat tubes in PCR machine with FINDNA program: 95ºC 15 min, 4ºC hold.
Add 20 µl of Tris (1 M, pH 8.0) to each tube, mix. Keep tubes at room temp.
Amplify
14.8 µl Water
2.0 µl 10x Std Pol Buffer
0.4 µl Rev primer (10µM)
0.4 µl Fwd primer (10µM)
0.2 µl dNTP (10 mM)
0.2 µl Taq (NEB)
2.0 µl Template
20 µl TOTAL
Amplify using CRISPRTQ program:
94º 2:00
94º 0:15 |
60º 0:15 | Repeat 35x
72º 0:45 |
72º 2:00
4º Hold
Mix 10 µl of PCR product with 1 µl loading buffer, and run on 2% gel to confirm successful amplification.
Some alleles may result in large insertions or deletions, but this is relatively uncommon. Most indels cannot be detected by PCR amplification with electrophoresis and will require either sequencing or restriction digest.
Mutation detection
Option 1: Sequencing
Send 5 µl (or remainder) of PCR product for Sanger sequencing with 1 µM fwd oligo.
Option 2: Specific PCR primers
If you're lucky enough to get a large deletion, primers spaced around the cut site may be able to reveal indels of ~25 bp or more. It may also be possible to design primers that will bind specifically to a mutant allele or WT allele. These could be used with a flanking primer to identify the exact genotype of a fish.
Option 3: Digest to detect mutated allele. This can be used if the predicted cut site is at a restriction site. However this will not necessarily result in destruction of the site in all mutant alleles.
Take finclip from fish at ≥4 weeks of age.
Add 180 µl of NaOH (50 mM) to each tube, keep at 95º for 10-15 minutes.
Heat tubes in PCR machine with FINDNA program: 95ºC 15 min, 4ºC hold.
Add 20 µl of Tris (1 M, pH 8.0) to each tube, mix. Keep tubes at room temp.
Amplify
14.8 µl Water
2.0 µl 10x Std Pol Buffer
0.4 µl Rev primer (10µM)
0.4 µl Fwd primer (10µM)
0.2 µl dNTP (10 mM)
0.2 µl Taq (NEB)
2.0 µl Template
20 µl TOTAL
Amplify using CRISPRTQ program:
94º 2:00
94º 0:15 |
60º 0:15 | Repeat 35x
72º 0:45 |
72º 2:00
4º Hold
Mix 10 µl of PCR product with 1 µl loading buffer, and run on 2% gel to confirm successful amplification.
Some alleles may result in large insertions or deletions, but this is relatively uncommon. Most indels cannot be detected by PCR amplification with electrophoresis and will require either sequencing or restriction digest.
Mutation detection
Option 1: Sequencing
Send 5 µl (or remainder) of PCR product for Sanger sequencing with 1 µM fwd oligo.
Option 2: Specific PCR primers
If you're lucky enough to get a large deletion, primers spaced around the cut site may be able to reveal indels of ~25 bp or more. It may also be possible to design primers that will bind specifically to a mutant allele or WT allele. These could be used with a flanking primer to identify the exact genotype of a fish.
Option 3: Digest to detect mutated allele. This can be used if the predicted cut site is at a restriction site. However this will not necessarily result in destruction of the site in all mutant alleles.