1. A CRISPR Approach to Gene Targeting
Dana Carroll 
Molecular Therapy 
Volume 20, Issue 9, Pages (September 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Consequences of targeted genomic cleavage. A double-strand break made by any type of cleavage reagent can be repaired by error-prone nonhomologous end joining (NHEJ), leaving small insertions and/or deletions at the site. An alternative mode of repair is homologous recombination (HR), which can use a manipulated donor DNA as a template, resulting in replacement of genomic sequences. The break can be made by any targetable nuclease: zinc-finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), homing endonucleases (HEs), or, perhaps, the new CRISPR reagents.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
ZFNs and TALENs. (Top) Each zinc finger (small ovals) in a zinc-finger nuclease (ZFN) binds primarily to three consecutive base pairs; a minimum of three fingers is required to provide sufficient affinity. Different colors indicate fingers recognizing different DNA triplets. Each set of fingers is joined to a FokI-derived cleavage domain (large ovals) by a short linker. (Bottom) In transcription activator–like effector nucleases (TALENs), each module (small ovals) binds a single base pair; the four colors indicate modules for each of the four base pairs. The minimum effective number of modules is 10–12, but more are typically used. The linker to the FokI domain (large ovals) is longer than for ZFNs and contains additional TALE-derived sequences.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
The type II CRISPR system. In a bacterial genome, identical repeats flank virus- or plasmid-derived spacer sequences in tandem arrays (blue). Long transcripts (green line) are processed into short RNAs containing a single spacer and a partial repeat. These short RNAs form partial duplexes with tracrRNAs and are bound by the Cas9 protein (orange oval). The complex then cleaves invading viral or plasmid DNA directed by the spacer RNAs. tracrRNA, trans-activated CRISPR RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
The CRISPR minimal-cleavage elements described by Jinek et al.1 A single RNA (green lines) with the critical elements of spacer and tracrRNA binds Cas9 protein (orange oval) and directs cleavage (arrowheads) to a sequence in DNA (blue) that has homology to the spacer. The region of RNA–DNA base pairing provides cleavage specificity. The target must also have a particular two– to three–base pair sequence adjacent to the region of homology, called PAM, which is recognized by the complex. PAM, protospacer adjacent motif; tracrRNA, trans-activated CRISPR RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions