Repair of ZFN-mediated cleavage by NHEJ and HDR pathways (Part 16- CRISPR in gene editing and beyond)
Welcome to the 16th part of the multi-part series on applications of CRISPR in gene editing and beyond.
Non-Homologous End Joining (NHEJ)
Non-Homologous End Joining is a rapid and efficient double-stranded repair mechanism that involves a simple ligation of the two DNA ends that result from a double-stranded break. The FokI-mediated DNA cleavage leaves overhanging ends, which can either be filled in completely or incompletely by the host polymerase enzyme or chewed back by limited exonuclease activity. Then the ligase enzyme ligates the two DNA ends to complete the repair process. As a result of this repair process, small insertions or deletions of nucleotides can be generated in the region, flanking the ZFN-mediated cleavage site.
When the zinc finger nucleases are targeted to the protein-coding regions, the outcome is often a shift in the reading frame, usually leading to a null allele or non-functional allele of the targeted gene. The zinc finger nucleases combined with repairing by non-homologous end joining can be used for introducing mutations or for knocking out a gene. Knocking out a gene means making the gene inactivate or inoperative. Let’s understand it with an example. In a study, researchers designed a pair of zinc finger nucleases targeted to the first coding exon of CCR5. CCR5 is the major co-receptor for HIV entry and is present on the human T cell. When these ZFNs were introduced into human T cell lines, the zinc finger nucleases disrupted the CCR5 gene. This ultimately resulted in decreased expression of CCR5, as well as protection from HIV infection because it blocked the entry of HIV into T cells.
Homology-directed repair (HDR) pathway
Alternatively, the homology-directed repair pathway, abbreviated as HDR, utilizes the presence of a template DNA for repairing double-stranded breaks. The template DNA carries the desired sequence of the gene of interest and is flanked by approximately 1 kbp homology on either side of the mutation. The template DNA then replaces the target double-stranded locus to restore genetic information in an error-free manner.
When ZFN binds to the targeted DNA, and the FokI enzyme introduces double-stranded breaks in it (Fig 4a), the introduction of double-stranded breaks activates the DNA damage repair by homology-directed repair. Therefore, after ZFN cleavage, the ends of the target DNA are digested by the 5′-3′ exonuclease enzyme, leaving 3' overhangs (Fig 4b). One of the resulting single-stranded 3′ ends invades homologous sequences in the template DNA (Fig 4c). The invading 3′ end is then extended by DNA polymerase represented by a dashed line (Fig 4d). After some synthesis, the extended end withdraws and anneals to the other end at the original break (Fig 4e). The gaps are filled in, and the continuity of the strands is restored by ligation (Fig 4f).
In a study, the researchers designed ZFNs targeting the IL2Rγ gene, which is mutated in X-linked severe combined immune deficiency syndrome or SCID. Specifically, the ZFNs were designed to target exon 5 of the IL2Rγ gene. To introduce the desired changes, a plasmid containing the unmutated or wild-type exon with approximately 1 kbp homology on either side of the mutation was introduced into the cells. Co-transfection of ZFNs and the plasmid carrying desired DNA into cells replaced the mutated IL2Rγ exon 5 with wild-type exon 5. Thus fixing the gene responsible for causing X-linked severe combined immune deficiency syndrome.
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