Biology Now, 2e

160 ■ CHAPTER 09 What Genes Are

GENETICS

of two RNA molecules that guide two Cas9

proteins to chosen, precise sites in a genome.

There, Cas9 efficiently cleaves both strands of

the DNA (Figure 9.6).

The CRISPR system is like a “Delete” key or,

in certain experiments, the cut-and-paste tool

of a word processing program—except with

nucleotides and genes instead of letters and

words. While most new laboratory tools can take

months or years to become widely used, CRISPR

was immediately a lab favorite, with genetic

studies that used the tool being published right

and left: editing the zebra fish genome, correct-

ing genetic disease mutations in adult mice,

enhancing pest resistance in wheat, and more.

Shortly after Doudna and Charpentier’s dis-

covery, both Feng Zhang (at the Broad Institute

of MIT and Harvard) and Church’s lab engi-

neered CRISPR systems for genome editing

in eukaryotic cells. With that innovation, the

Church team was ready to start editing the pig

genome, but first they needed to identify how

many copies of the PERV DNA the genome

contained. Using laboratory tools to sequence

the pig DNA, they identified 62 copies of PERVs

scattered throughout the genome.

Sixty-two may sound like a lot, but it’s a

drop in the bucket compared to the number of

genes in a mammalian genome. Pigs have over

1,600 genes for their sense of smell alone. An

estimated 19,000 protein-coding genes are

packed into the human genome, surrounded

by even more DNA that does not code for

proteins. Cells are adept at stuffing an enor-

mous amount of DNA into a small space; they

use a variety of packaging proteins to wind,

fold, and compress the DNA double helix, going

through several levels of packing to create the

DNA-protein complex that we call a chromo-

some (Figure 9.7).

Short lengths of double-stranded DNA are

wound around “spools” of proteins, known as

histone proteins, to create a beads-on-a-string

structure consisting of many histone beads,

called nucleosomes, connected by strings

of DNA. This beads-on-a-string structure is

compressed and coiled into a more compact

form, known as the chromatin fiber, by yet

other types of packaging proteins. The chroma-

tin fiber is then looped back and forth to further

condense and coil again to form the resulting

chromosome in the interior of the nucleus.

1

2

3

4

PERV DNA

Guide RNA

Cas9

Guide RNA

The CRISPR-Cas9

system is made up of

guide RNA molecules

and Cas9 proteins.

pol

The pol gene within a

double-stranded

PERV DNA sequence

is targeted within the

pig genome. The

guide RNAs bring

Cas9 to their target

DNA sequences via

complementary

base-pairing.

Cas9 cuts through

both strands of the

target DNA in the pig

chromosome, and

the CRISPR-Cas9

complex is released.

During normal repair

processes, the DNA

sequences between

the cut sites are

removed and the

remaining DNA is

relinked, “knocking

out” most of the pol

gene. What remains

of pol is not

functional.

Figure 9.6

Genome editing with CRISPR-Cas9, an efficient and

cost-effective tool

Q1: What common mechanism is employed by the guide RNA to find

its target DNA sequence?

Q2: How many strands of DNA must Cas9 cut to be effective?

Q3: Does Cas9 also cause the deletion of DNA from the genome?