6
These endonucleases are highly efficient and have been used in the modification of
human cells as well as numerous other eukaryotic systems [ 16 , 17 ]. However, rapid
design and selection of ZFNs with novel binding sites that do not cleave off-target
regions can still be a challenge [ 12 , 18 ].
Inspired by the success of ZFNs, many groups turned their attention to DNA bind-ing transcription activator-like (TAL) proteins of Xanthomonas, a plant pathogen that
uses these domains for virulence factors in the nuclease of their host plant [ 19 , 20 ].
TAL proteins, like zinc fingers, have a motif code for binding to DNA nucleotides,
this time with a repeat motif recognizing one nucleotide instead of three, as in the
case of zinc finger proteins [ 19 , 20 ]. Fusion of an endonuclease, such as FokI, to
these TAL proteins created TAL endonucleases or TALENs that offered another tool
for engineering genomes [ 21 , 22 ]. These proteins, while efficient at stimulating HR,
have a downside of being large and contain repetitive regions, which can be problem-
atic on the DNA level due to mutagenic recombination events [ 12 , 23 ].
Fig. 1.2 Schematic of CRISPR-Cas9 DNA cleavage and DNA repair. (a) Cas9 complexes with
the sgRNA to direct cleavage to region specified by the target sequence encoded on the sgRNA. (b)
Repair of the double-stranded cut can occur via non-homologous end joining or homologous
recombination. In the case of homologous recombination, if a donor DNA is supplied exogenously
the region can be modified in a targeted fashion
J.E. DiCarlo et al.