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BIOCHEmISTRY ``BIOCHEMISTRY—lABORATORY TECHNIqUES BIOCHEmISTRY ``BIOCHEMISTRY—lABORATORY TECHNIqUES SECTION II 53

CRISPR/Cas9 A genome editing tool derived from bacteria. Consists of a guide RNA (gRNA) , which is
complementary to a target DNA sequence, and an endonuclease (Cas9), which makes a single-
or double-strand break at the target site . Break imperfectly repaired by nonhomologous end
joining (NHEJ) Ž accidental frameshift mutations (“knock-out”) , or a donor DNA sequence
can be added to fill in the gap using homology-directed repair (HDR) .
Not used clinically. Potential applications include removing virulence factors from pathogens, replacing
disease-causing alleles of genes with healthy variants, and specifically targeting tumor cells.

Cas9 gRNA

Donor DNA
NHEJ 3 A 3 B HDR
+

Frameshift/inactivation Edited sequence
(”knock-out”) (”knock-in”)

Blotting procedures
Southern blot 1. DNA sample is enzymatically cleaved into I: Parentsrentsrents
I: Pa
I: Pa
smaller pieces, which are separated on a gel
by electrophoresis, and then transferred to a PEDIGREE PEDIGREE PEDIGREE
filter.
II: Childr
II: Childr
2. Filter is exposed to radiolabeled DNA II: Childrenenen
probe that recognizes and anneals to its Aa Aa aa Aa AA Genotype
Genotype
Genotype
complementary strand.
3. Resulting double-stranded, labeled piece of Mutant
Mutant
Mutant
DNA is visualized when filter is exposed to SOUTHERN BLOT SOUTHERN BLOT SOUTHERN BLOT Normal
Normal
Normal
film.
Northern blot Similar to Southern blot, except that an RNA
sample is electrophoresed. Useful for studying SNoW
mRNA levels, which are reflective of gene DRoP:
expression. Southern = DNA
Northern = RNA
Western blot Sample protein is separated via gel electrophoresis
and transferred to a membrane. Labeled Western = Protein
antibody is used to bind to relevant protein.
Southwestern blot Identifies DNA-binding proteins (eg, c-Jun,
c-Fos [leucine zipper motif]) using labeled
double-stranded DNA probes.

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