BIO421
Advanced Genetics
Lecture 13
Genome Editing
I. Why should we edit the genome?
A. To induce mutations that are molecular nulls
1. These mutations eliminate the production of the protein or severely alter it (stop or nonsense codon alterations, for instance)
B. To perform reverse genetics by knocking out a gene whose sequence suggests it is involved in a
pathway of interest to us.
C. To knock in sequences such as reporters.
II. Old-time protocols
A. Random mutagenesis for deletions with PCR based assay for specific genes
B. Transposons
1. Use of transposons that lack a functional transposase allow transposition under the
experimenter’s control
2. Once a useful transposon insertion has been found, its genomic location is easily identified
via PCR.
III. Newer protocols that induce double strand breaks
A. Zinc-finger nucleases
1. Utilize zinc-finger motifs from common transcription factors
a. These can be designed to recognize nearly any sequence.
b. Each zinc-finger repeat recognizes 3 base pairs.
2. Combined with the endonuclease domain from the Fok1 restriction enzyme
3. Drawbacks:
a. not guaranteed that engineered zinc-fingers will actually bind sequence desired
b. large risk of off-site binding and DNA damage
B. TALENs (Transcription Activator-like Effector Nucleases)
1. Combines the TALE DNA binding domain with the Fok1 nuclease
2. Easier to design the TALE DNA binding domains, and they are more likely to bind targeted
site and less off-site damage
C. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats)
1. Discovered as a bacterial immune system
a. The palindromic repeats separate captured sequences from invading nucleic acids, like
viral genomes and plasmids
b. The CRISPR-Cas system destroys invading nucleic acids that match those in the
captured sequences.
2. The CRISPR-Cas system and been engineered to create double strand breaks (DSBs).
a. The researcher needs to design a single guide RNA that targets a sequence with a PAM
site (NGG).
b. The researcher then transforms their organisms with the Cas9 sequence and the single
guide RNA sequence, and a DSB will be made at the proper site near the PAM
sequence.
c. Base pairing between the sgRNA and the target sequence provides the specificity
d. Much easier to work with than Zinc-finger nucleases or TALENs
D. So what do we do with double strand breaks
1. Allow them to self repair by non-homologous end joining (NHEJ), which frequently results
in an indel (insertion or deletion)
2. Provide a template for repair by homologous recombination. By this method, we can insert
novel sequences into the double strand break, like reporter constructs.