Design a mutation in any gene of a mammalian or plant of your choice*. In order to design a genomic modification you will need to know the gene sequence you want to modify. At the end of the document is a short selection of organisms with published genomes, but you can also take the gene sequence of the organism you want to work in from elsewhere.
1.) Overview and rationale: In a short paragraph, describe the type of mutation you want to introduce and the rationale behind it. For inspiration you can take a look at Prof. Church’s list of potential human genome modifications, browse in one of the functional data bases, or look through published experiments.
2.) Genomic sequence: Include the genomic sequence you want to modify in your write up. You don’t need to paste the full sequence; just include the part relevant for designing your editing experiment!
3.) Genome editor design: Describe which genome editing tool you want to use and submit your design.
For CRISPR/Cas9-based editors this means that you will include a sequence of your guide RNA and the name of the Cas9 protein you want to use. SpCas9 is most commonly used and recognizes an 5’-NGG-3’ PAM site (where ‘N’ can be any base), but you might want to use another protein with different PAM sequence for your experiment.
In addition, if you are not designing a gene knockout (introducing a double strand break into the DNA, followed by NHEJ repair), but an HDR experiment, include the sequence of the repair template which allows you to achieve your desired mutation.
Challenge: If you are already experienced in designing mutations with CRISPR/Cas9 or want to XX use another editor, we have resources and publications listed below and don’t hesitate to reach out to the teaching fellows! Some additional genome engineering tools:
o TALENs: submit amino acid sequence/DNA sequence encoding the protein
o ZFNs: submit amino acid sequence/DNA sequence encoding the protein
o Base editors: submit gRNA
o Prime editors: submit gRNA
Link to editors and tutorials
Base editors contain a nicking or dead Cas9 enzyme fused to a deaminase.
a.) PAM requirement: Base editors contain a nicking or dead Cas9 enzyme fused to a deaminase. For designing your guide RNA for base editing you will therefore have a PAM requirement like you would have for any Cas9 experiment.
b.) Deamination window: An additional design constraint is that the sequence window in which deamination occurs is only a few base pairs long. You can find information on the deamination windows in the review below (even though some new editors are not included).
BE4 and ABE7.10 are good starting points and both use SpCas9 with NGG Pam requirement. Base editors with other PAM sites have been constructed too.
For TALENs, you can assume no sequence restrictions -- One of the technology’s previous restrictions was a T starting base, but this has since been overcome. In contrast to the CRISPR/Cas technologies above, your DNA sequence is recognized through interactions between the DNA and the TALEN: each TAL in the array recognizes one base.
Note: In order to introduce a double strand break, you will need to design to TALENs targeting the opposing strands.
Protocol: modified from https://www.wired.co.uk/article/extract-dna
Required background reading:
The ABO gene encodes the ABO blood group glycosyltransferase. This enzyme catalyzes the transfer of a sugar group to the H-antigen. In people with blood group O, the ABO glycosyltransferase is inactive, due to a single base deletion in the allele (a frameshift mutation). Meanwhile, the A and B allele differ by several base substitutions which alter the sugar groups transferred by the enzyme: in people with blood group A, N-acetylgalactosamine is transferred; while for blood group A, the enzyme transfers galactose.
Lab (performed by TA):
Additional background lecture (optional): https://www.youtube.com/watch?v=YnF1b_Kqf88