As an example, for correction of shorter sequences single-stranded oligonucleotide donors (ssODNs) facilitate greater editing efficiency than double-stranded donors. The required length of these homology arms, as well as the structural nature of the template DNA itself is determined largely by the size of the desired insert. This scaffold must contain a nucleic acid segment complementary to the desired mutation and flanked by homology arms (complementary sequences to either end of the DSB) that localize the template to the correct genomic sequence. 13 This mechanism of repair involves the incorporation of a DNA template strand as a homologous scaffold. 12 Alternatively, the incorporation of a homologous ‘repair template’ DNA strand along with the CRISPR/Cas9 machinery provides the basis for HDR. Because this random incorporation can frequently lead to frame-shift mutations, this method can be used to knock-out gene expression. The principle of NHEJ involves the incorporation of random nucleotides at the break site, until a small amount of strand overlap allows DNA polymerase enzymes to re-joint the strands. knock-out), activity of the Cas9/sgRNA complex alone is sufficient, and as such repair typically occurs through the NHEJ pathway. 11 Repair after the DSB can occur through two main mechanisms, non-homologous end-joining (NHEJ) and homology-directed repair (HDR). 6 Depending on the desired alteration, different mechanisms for repair are utilized, with results that can be categorized as a) gene disruption, b) gene correction, c) gene insertion, and d) large gene deletion ( Figure 1).
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Endogenous cellular repair mechanisms typically repair the break, using a series of polymerases and ligase enzymes to add or remove nucleotides to the break point before re-joining the broken strands. In the CRISPR editing process a DSB is generated by Cas9. The discovery that a single sgRNA molecule could replace these two molecules greatly simplified the CRISPR/Cas9 system, and today Cas9-mediated DNA cleavage can be obtained with high sequence specificity based on the facile redesign of sgRNA alone. In nature, most Cas9 variants, including spCas9, require two separate RNA molecules for their targeted nuclease activity CRISPR-RNA (crRNA) and trans-activating crRNA (tracrRNA) are both required in order to form a functional Cas9-sgRNA ribonucleoprotein complex (Cas9-RNP). Of these systems, the most widely used Cas9 nuclease is derived from Streptococcus pyogenes and expressed in E. These variants feature significantly different properties, including size, targeting sequence (protospacer-adjacent motif, or PAM) specificity, and the location where double stranded break (DSB) occurs. 8 Several Cas9 nuclease variants have been identified from different bacterial strains. 7 CRISPR/Cas9 systems are originally a component of the bacterial innate immune system, where they provide a ‘memory’ platform for the host’s immune response to effectively counter repeated viral infection. The other element is single-guide RNA (sgRNA) molecules that complex with the Cas9 protein and guide it to its genomic site of action by forming complementary base pairing with the target sequence. The first component is the Cas9 protein, an endonuclease capable of double-stranded cleavage of DNA. The CRISPR/Cas9 system is composed of two main elements. These strategies benefit from flexible yet high-fidelity sequence targeting and efficacious editing, underscoring their position as perhaps the foremost technique for mammalian gene editing.
CRISPR/Cas9 systems are simple, elegant solutions for selective alteration and interrogation of the genome. 3, 4 Clustered regularly-interspaced short palindromic repeat (CRISPR)-related systems have become a cornerstone of current gene editing-based approaches for curing human disease. 2 While effective, these techniques notoriously require extensive design when choosing new genetic targets and can suffer from barriers to fidelity and efficiency of editing. 1 In recent years, gene-based approaches to disease therapy have gained significant traction in research, as evidenced by the emerging interest in gene editing technologies including meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Despite the fact that over 7000 identified diseases have been linked to alterations in the human genome, effective treatments have only been developed for ~500.
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