1. Chemical Biology Approaches to Genome Editing: Understanding, Controlling, and Delivering Programmable Nucleases 
Johnny H. Hu, Kevin M. Davis, David R. Liu 
Cell Chemical Biology 
Volume 23, Issue 1, Pages (January 2016)
DOI: /j.chembiol
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2. Figure 1
DNA Cleavage by Zinc-Finger Nucleases, Transcription Activator-like Effector Nucleases, and the Clustered Regularly Interspaced Short Palindromic Repeat-Cas9 System
(A) Once a programmable nuclease catalyzes double-stranded DNA cleavage, endogenous cellular non-homologous end-joining (NHEJ) or homology-directed repair (HDR) processes mediate DNA modification at the cleaved locus. While NHEJ yields small, random insertions or deletions (indels) at the cleavage site, HDR uses a separate single- or double-stranded DNA molecule as a template for the precise replacement of bases spanning the site of cleavage.
(B) Zinc-finger nucleases contain repeats of zinc-finger domains, each of which recognizes 3 bp of DNA. By fusing the DNA-binding domain to a FokI nuclease, a dimeric ZFN recognizes and cleaves DNA containing adjacent left and right half-sites.
(C) TALENs consist of an array of 33- to 35-amino acid TALE domains fused to FokI nuclease. DNA specificity is determined by two amino acids, known as the repeat-variable di-residues (RVDs), within each TALE domain. By simply linking TALE modules together, TALENs can be programmed to recognize virtually any DNA sequence. Like ZFNs, TALENs are designed to recognize a left and right half-site. Upon DNA binding, FokI domain dimerization enables double-stranded DNA cleavage.
(D) The CRISPR-Cas9 system is an RNA-programmed endonuclease. The guide RNA (gRNA) contains both the sequence necessary to bind to the Cas9 protein and a “spacer” sequence that binds to the target DNA following Watson-Crick base-pairing rules. By altering the spacer sequences in the gRNA, Cas9 can be targeted to virtually any DNA sequence that contains a short protospacer adjacent motif (PAM) downstream of the DNA target. The most commonly used S. pyogenes Cas9 makes a double-stranded cut 3–4 bp away from the PAM as indicated by the arrowheads.
Cell Chemical Biology  , 57-73DOI: ( /j.chembiol )
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3. Figure 2
Improving Cas9 Specificity through Protein and Guide RNA Engineering
(A) The D10A mutation in S. pyogenes Cas9 converts the protein from a DNA nuclease to a DNA nickase (Cas9n) with the ability to cleave only the strand complementary to the gRNA. A pair of Cas9n-gRNA nickases programmed to nick opposite strands at nearby locations as indicated by the arrowheads can induce double-stranded breaks that leads to NHEJ and HDR at levels similar to that of wild-type Cas9, but with improved specificity due to the requirement of two adjacent nicking events.
(B) By fusing FokI to a catalytically dead Cas9 (fCas9), DNA cleavage only occurs when two fCas9 monomers assemble at adjacent DNA sites to form a catalytically active FokI nuclease.
(C) Compared with a canonical single-guide RNA (sgRNA) that contains 20 nucleotides complementary to the target, trugRNAs contain shortened regions of target complementarity of 17–19 nucleotides while ggX20-gRNA adds two extra mismatched guanine nucleotides to the 5′ end of the gRNA. Both changes can increase the specificity of Cas9:gRNA-mediated DNA cleavage.
Cell Chemical Biology  , 57-73DOI: ( /j.chembiol )
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4. Figure 3 Small-Molecule-Controlled Cas9 Variants
(A) A laboratory-evolved intein that only undergoes protein splicing in the presence of the cell-permeable small molecule 4-hydroxytamoxifen (4-HT) was inserted into Cas9. The presence of the intein disrupts Cas9 activity until treatment with 4-HT, which induces protein splicing and restores Cas9 activity.
(B) After splitting Cas9 into an N-terminal fragment and a C-terminal fragment, an FKBP rapamycin binding (FRB) domain or FK506 binding protein (FKBP) was fused to the N-terminal fragment or C-terminal fragment, respectively. To decrease background activity, a nuclear export signal (NES) was added to the N-terminal fragment while two NLS signals were added to the C-terminal fragment. The addition of rapamycin induces dimerization of Cas9(N)-FRB-NES and Cas9(C)-FKBP-2xNLS, resulting in trafficking of the reassembled Cas9 protein to the nucleus, where it is able to target and cleave DNA.
Cell Chemical Biology  , 57-73DOI: ( /j.chembiol )
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5. Figure 4 Light-Controlled Cas9 Variants
(A) A photoactivatable Cas9 (paCas9) was created by fusing the photoswitchable proteins pMag or nMag, to the N-terminal fragment or C-terminal fragment, respectively, of a split Cas9 protein. Upon blue light irradiation, dimerization of pMag and nMag leads to activation of Cas9 and cleavage of target DNA.
(B) In the light-activated CRISPR-Cas9 effector (LACE) system, a truncated version of CIB1 is fused to the N and C termini of Cas9, and CRY2 is fused to the transcriptional activation domain VP64. In the presence of blue light, CIB1 and CRY2 binding leads to localization of VP64 to the site of Cas9 binding and transcriptional activation.
Cell Chemical Biology  , 57-73DOI: ( /j.chembiol )
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6. Figure 5
Intracellular Delivery of TALE and Cas9 Proteins Using Cationic Lipids
(A) Proteins fused to supernegatively charged proteins such as (-30)GFP, or Cas9 protein complexed with gRNA, acquire a high degree of anionic charge.
(B) Cationic lipids commonly used to transfect DNA and RNA can complex with the resulting highly anionic proteins or protein:RNA complexes, mediating their potent delivery into mammalian cells.
Cell Chemical Biology  , 57-73DOI: ( /j.chembiol )
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