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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Nat Neurosci. 2021 Feb 1;24(3):297–311. doi: 10.1038/s41593-020-00778-1

Figure 2. Mechanisms of genome editing by engineered nucleases.

Figure 2.

a) Double-strand breaks (DSBs, arrowhead) in genomic DNA are repaired by non-homologous end joining (NHEJ), that introduces indel mutations (red dash lines), typically leading to a change of the reading frame, pre-mature termination codon (PTC), and disrupted gene function.

b) Alternately, in the presence of donor template, homology-directed repair (HDR) mechanisms lead to precise insertion or modification of DNA (green lines).

c) Zinc finger nucleases (ZFNs) are composed of three to six zinc finger domains (color circles), each recognizing three nucleotides. The zinc finger domains on opposite strands of DNA bring two Fok1 endonuclease domains together, inducing Fok1 dimerization and DSB.

d) Transcription activator-like effector nucleases (TALENs) have 16-20 TALE monomers, each recognizing a single nucleotide. A pair of TALENs induces Fok1 dimerization and DSB.

e) The CRISPR-Cas9 system contains a synthetic guide RNA and the nuclease Cas9. The guide RNA has two domains – a programmable crRNA sequence recognizing the genomic target, and a tracrRNA sequence for Cas9-binding.

f) Location of PTCs determine transcription fate. Only PTCs residing >50-55 nt upstream of the last exon-exon junction will lead to non-sense mediated decay (NMD) and degradation of the mRNA. The last exon is exempt from NMD, and PTCs in this “NMD-insensitive region” should lead to truncations (see article for details).