NHEJ typically results in insertions/deletions (indels), which may lead to disruption of target gene function (4).ĬRISPR/Cas9-mediated gene disruption - principle and components. In the absence of donor DNA, the DSB is repaired by the non-homologous end joining (NHEJ) DNA repair pathway, which is an error-prone process. The combination of CRISPR and Cas9 results in a double-stranded break (DSB) at the target DNA site (3). The requirement for a PAM sequence limits the number of possible target sequences, although from a practical standpoint, there are usually sufficient numbers of possible targets within a gene to design CRISPRs for appropriate gene knockout. Cas9 induces DNA breaks 3bp upstream from a proto-spacer adjacent motif (PAM) sequence adjacent to the target sequence, consisting of 5′-NGG-3′ motif (2). As outlined in Figure 1, a typical CRISPR construct consists of a single guide RNA that includes a 20-nucleotide sequence specific to the DNA target and an 80-nucleotide Cas9 interaction loop (1) that binds to a recombinant form of Cas9 protein. The CRISPR/Cas9 system takes advantage of the ability of Cas9 endonuclease to cleave DNA by directing cleavage to specific targeted regions of the genome. This Unit describes the fundamentals of CRISPR/Cas9 technology and its application to high-throughput genetic screens in zebrafish in the context of a detailed methodological approach for the practical application of CRISPR/Cas9 techniques. The advent of CRISPR/Cas9 based genome editing in 2012 was a huge step forward in addressing the shortcomings of the morpholino approach ( Jinek et al., 2012). However, morpholino-based approaches have drawbacks such as lack of germ line transmission, and the effects of morpholino knockdown are both transient and restricted to early developmental stages.

The introduction of of morpholino-based gene knock down approaches revolutionized the use of zebrafish as a model system to perform large-scale in-vivo, reverse genetic screens. Prior to the advent of CRISPR/Cas9 technology, the methods of choice for reverse genetics analysis of specific gene function in zebrafish were less efficient genome editing technologies or a morpholino-based gene knockdown ( Nasevicius & Ekker, 2000). Combining the power of CRISPR/Cas9 technology with the zebrafish as a well-defined model organism has transformed the field of developmental biology, the study of human genetic diseases and the application of high-throughput genomic screens. The zebrafish ( Danio rerio) is uniquely suited to study gene function, due to its rapid development rate, the ability to obtain large number of embryos, the transparent nature of embryos in the first 48 hours and its diploid status. Clustered regularly interspaced small palindromic repeats (CRISPR)/Cas9 based genome editing tools are extensively used for generating mutations in target genomic regions in model systems ranging from cell lines to model organisms.

Ape sequence analysis