The demand for targeted, fast, and customizable gene knockout has rapidly grown over the last decade. The molecular basis for gene editing relies on the cellular repair mechanisms of DSBs. These lesions can be repaired by HRR or NHEJ as mentioned above (Takata et al., 1998) (see 1.2.2.3). HRR is an error-free process, but the cell depends on a template strand and therefore it only takes place in S, G2, or M phase of the cell cycle (reviewed in Szostak et al., 1983). DSBs are re-ligated through the NHEJ pathway in the absence of a repair template, resulting in insertion/deletion mutations leading to frameshift mutations and premature stop codons (Deltcheva et al., 2011). For genome editing it is critical to introduce a directed DSB in the target gene. Recently, notable attention has been paid to genome editing tools like zinc-finger nucleases (ZFNs), meganucleases or bacterial transcription activator-like type III effector nucleases (TALENs). Those techniques have been used for targeted gene knockout (KO) in multiple fields (Wood et al., 2011).
1.5.1 TALEN, Zinc finger and meganucleases
ZFNs or TALENs function through tethering of endonuclease catalytic domains to modular DNA-binding proteins to induce targeted DSBs at specific genomic loci (Wood et al., 2011).
ZFNs are artificial restriction enzymes in which a zinc-finger DNA-binding domain is fused to a DNA-cleavage domain (Kim et al., 1996). In order to cleave DNA, the cleavage domain has to dimerize and thus one pair of ZFNs is required to target non-palindromic DNA sites (Bitinaite et al., 1998).
TAL effectors are naturally occurring proteins from Xanthomonas, a plant bacterial pathogen.
The central targeting domain contains a series of 33–35 aa repeats each recognizing a single base pair (bp). Specificity is determined by two hypervariable aa, the repeat-variable diresidues (RVDs) (Deng et al., 2012; Mak et al., 2012). Modular TALE repeats are linked together to recognize contiguous DNA sequences, similar to ZFNs. In contrast to ZFN proteins, no re-engineering of the linkage between repeats is necessary to construct long arrays of TALEs with the ability to basically target single sites in the genome. DNA binding domains can be coupled to various effectors, most importantly including cleaving domains of nucleases like FokI to induce targeted DSBs (Cermak et al., 2011; reviewed in (Gaj et al., 2013). On the other hand, meganuclease technology utilizes naturally occurring homing endonucleases, e.g. I-CreI and I-SceI enzymes, to re-engineer their DNA-binding specificity to target novel sequences (reviewed in Chevalier & Stoddard, 2001).
Meganuclease and TALEN based gene editing approaches have already been used in preclinical trials for XP gene therapy. For example, Dupuy et al. (Dupuy et al., 2013) applied engineered meganuclease and TALEN to perform a targeted correction of an XPC mutation in a patient cell line (XP4PA). After successful XPC gene correction, the full-length XPC protein was re-expressed resulting in full recovery of WT UV resistance and functional repair capabilities in the patient cell line.
1.5.2 CRISPR/Cas9
Notwithstanding, the above mentioned techniques are often inefficient, time consuming, laborious, and expensive (Wei et al., 2013). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated (Cas) nuclease 9 system is adapted from the adaptive bacterial immune system of Streptococcus pyogenes that evolved in bacteria to defend against invading plasmids and viruses. In this setting, a nuclease is guided by small RNAs to highly efficiently target specific DNA sequences (Cong et al., 2013; Li et al., 2013b). In the CRISPR/Cas9 strategy, a DSB is induced by Cas9 cleavage and the target locus is typically repaired by the error-prone NHEJ or the error-free HRR. In the absence of a repair template, the NHEJ pathway re-ligates DSBs, resulting in insertion/deletion mutations
and therefore in frameshift mutations and premature stop codons as mentioned above (Deltcheva et al., 2011) (see 1.2.2.3).
In the natural context, invading DNA from viruses or plasmids is cut into small fragments and incorporated into the CRISPR locus amidst a series of short repeats (around 20 bps). When the loci are transcribed, and transcripts are processed, small RNAs (crRNA – CRISPR RNA) are generated guiding the Cas9 effector endonuclease together with a tracrRNA to target invading DNA based on sequence complementarity. Upon another encounter, the Cas9 complexed with a crRNA and separate tracrRNA then cleaves the foreign DNA containing the 20 nt crRNA complementary sequence adjacent to the PAM sequence. (Barrangou et al., 2007; Jinek et al., 2012) (see Figure 10).
Figure 10: Schematic overview of the CRISPR/Cas9 system of Streptococcus pyogenes
Foreign invading DNA (A) is incorporated into the bacterial genome at the CRISPR loci during the acquisition phase (B). The CRISPR loci is transcribed, and processed into crRNA during crRNA biogenesis (C). If the cell is challenged with the same foreign invading DNA again, the Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20 nt crRNA complementary sequence adjacent to the PAM sequence (D). Illustration by Dr. rer. nat. Christina Seebode.
For genome editing, synthetic guide RNAs have been simplified by fusing together the crRNA and tracrRNA of the CRISPR/Cas9 system (Jinek et al., 2012). This system can be applied to all tissues and cell types containing the mentioned DSB repair pathways. The advantage of the CRISPR/Cas9 system over ZFNs and TALENs lies in the markedly easier design, high specificity, efficiency, and applicability for high-throughput and multiplexed gene editing in a variety of cell types and organisms bringing the CRISPR/Cas9 technology to the fore of the genome editing field (reviewed in Boettcher & McManus, 2015).
1.5.3 Limitations and precautions
Special attention has to be paid to nuclease off-target activity, which can be predicted by several online tools (Hsu et al., 2013; Pellagatti et al., 2015). However, a precise prediction of off-target sites due to tolerable mismatches, epigenomic effects, DNA methylation and chromatin structure is not possible yet. However, off-target mutations are less common in CRISPR/Cas9 systems compared to ZFNs, TALENs, and meganucleases analyzed by whole-genome sequencing studies (Suzuki et al., 2014; Veres et al., 2014). In regard to cleavage efficiency, it has to be mentioned that ZFNs show relatively weak cleavage of chromosomal DNA, in comparison to 100% cleavage efficiency of TALENs in mammalian cell lines (Kim et al., 2010). Concededly, mismatched dimer formation of TALENs can cause high mutation rates (Kim et al., 2013). Interestingly, ZFNs are nearly equally effective in creating both deletions and insertions, while TALENs preferably induce deletions (Cornu et al., 2008).