Essential Cell Biology 5th edition

358 CHAPTER 10 Analyzing the Structure and Function of Genes
Genes Can Be Edited with Great Precision Using the
Bacterial CRISPR System
Bacteria employ several mechanisms to protect themselves from foreign
DNA. One line of defense is provided by the restriction enzymes, as previously
discussed. Recently, the discovery of another bacterial defense
system led to the development of a powerful new method for editing
genes in a variety of cells, tissues, and organisms. This system, called
CRISPR, relies on a bacterial enzyme called Cas9, which produces a double-strand
break in a molecule of DNA. Unlike restriction enzymes, Cas9
is not sequence-specific; to direct Cas9 to its target sequence, investigators
provide the enzyme with a guide RNA molecule. This guide RNA,
carried by Cas9, allows the enzyme to search the genome and bind to a
segment of DNA with a complementary sequence (Figure 10−31A). The
gene coding for Cas9 has been genetically engineered into a variety of
organisms; thus, to use the CRISPR system to target a gene—or multiple
genes—researchers need only introduce the appropriate guide RNAs
(Movie 10.2).
As we saw in Chapter 6, double-strand breaks, like the one induced by
Cas9, are often repaired by homologous recombination—a process that
uses the information on an undamaged segment of DNA to repair the
break. Thus, to replace a target gene using CRISPR, investigators simply
provide an altered version of the gene to serve as a template for the
homologous repair. In this way, a target gene can be selectively cut by
the CRISPR system and replaced at high efficiency by an experimentally
altered version of the gene (Figure 10−31B).The CRISPR system therefore
provides another means of generating transgenic organisms.
Researchers are also adapting the CRISPR system for turning selected
genes on or off. In this case, a catalytically inactive Cas9 protein can be
3′
guide RNA
Figure 10–31 The CRISPR system can be
used to study gene function in a variety
of species. (A) The Cas9 protein, along with
a guide RNA designed by the experimenter,
are both artificially expressed in the cell or
species of interest. One portion of the guide
RNA (light blue) associates with Cas9, and
another segment (dark blue) is designed to
match a particular target sequence in the
genome. (B) Once Cas9 has made a doublestrand
break in the target gene, that gene
can be replaced with an experimentally
altered gene by the enzymes that repair
double-strand breaks through homologous
recombination (see Figure 6−31). In this way,
the CRISPR system promotes the precise
and rapid replacement of a target gene.
(C and D) By using a mutant form of Cas9
that can no longer cleave DNA, Cas9 can
be used to activate a normally dormant
gene (C) or turn off an actively expressed
gene (D). (Adapted from P. Mali et al., Nat.
Methods 10:957–963, 2013.)
Cas9
protein
3′
5′
(A)
double-strand break
made by Cas9
(B)
catalytically inactive Cas9 fused
with transcription activator
(C)
upstream
recognition
sequence
target gene
TARGET
GENE ON
cleavage site
cleavage site
altered version of
target gene produced
by genetic engineering
HOMOLOGOUS
RECOMBINATION
double-stranded
DNA in genome
target gene replaced
by altered version
catalytically inactive Cas9 fused
with transcription repressor
(D)
TARGET
GENE OFF