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E. González-Romero et al.
(TALEN),5 opening new horizons for genome manipula- tion (Figure 1A). Nevertheless, designing the aforemen- tioned nucleases to induce DSB in specific loci relies on predicting protein-DNA interactions, which remains technically challenging, and so these nucleases are not practicable in every laboratory. By contrast, the recent breakthrough of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technology, which is based on nucleic acid interactions, has enabled specific genome editing in a versatile and uncomplicated manner over previous nucleases, and has
A
revolutionized the field of genome engineering (Figure 1B, Table 1).
CRISPR sequence repeats were first reported in Escherichia coli6 and were later characterized in Haloferax mediterranei, an archaeon isolated from a hypersaline envi- ronment in Alicante (Spain).7 Soon after, these sequence repeats were identified as a part of a primitive adaptive immune system in prokaryotes.8,9 In 2012, Doudna and Charpentier demonstrated the first use of CRISPR/Cas9 to introduce site-specific DSB in target DNA based on the ability of a single guide RNA (gRNA) to direct sequence-
Figure 1. Nucleases used in genome engineering. (A) Pre- CRISPR nucleases such as meganu- cleases, zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are pro- teins that bind directly to DNA. Meganucleases are naturally occur- ring restriction enzymes that recog- nize between 12 to 40 base pair sequences, although they allow for some restricted level of engineering to make them specific to certain loci. Engineered ZFN induce specific double-strand breaks (DSB) acting as dimers. Each monomer is com- posed of a non-specific cleavage domain from the FokI endonuclease and a zinc-finger protein array where each domain bind three base pairs. ZFN dimers are able to recog- nize 18–24 base pairs in the target sequence, allowing for highly specif- ic targeting. TALEN are designed combining the same non-specific endonuclease FokI domain and transcription activator-like effector (TALE) proteins. TALE proteins pres- ent a central domain responsible for DNA binding, which interacts specif- ically with just one nucleotide. One of these domains consists of monomers of 34 amino acid residues, two of which are responsi- ble for nucleotide recognition. This makes the design of TALEN very straightforward in principle. (B) In contrast to the nucleases described in (A), the Cas9 endonuclease of the CRISPR/Cas9 system binds to the target DNA thought the guide RNA (gRNA) by Watson-Crick base pair- ing. The gRNA is composed of two molecules of RNA: (i) the CRISPR RNA (crRNA) (green nucleotides) of which 20 nucleotides [white bold in top panel in (A), black bold in middle and bottom panels in (A)] show strict homology to the target and (ii) the trans-activating crRNA (tracrRNA), which binds to the crRNA and to the Cas9 nuclease (yellow structure). The gRNA brings Cas9 the target sequence, which is always adjacent to a protospacer adjacent motif (PAM) sequence. The PAM sequence for the most used Cas9, isolated from the bacteria Streptococcus pyogenes, is NGG (TGG in the white box). Notes: white arrows in (A) represent hydrogen bonds between amino acids from proteins and DNA base pairs; thick black arrows point to the site of cleavage of the nucleases.
B
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haematologica | 2019; 104(5)