The ability to make specific changes to DNA, such as changing, inserting or deleting sequences that encode proteins, enables researchers to engineer cells, tis­sues and organisms for practical applications. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adaptive immune system effector, has been shown to facilitate RNA-guided site-specific DNA cleavage in bacteria, suggesting a simple alternative strategy for genome engineering (Sorek et al. 2013). The CRISPRs are a diverse family of DNA repeats that all share a common architecture. Each CRISPR locus consists of a series of short repeat sequences (typically 20-50 bp long) separated by unique spacer sequences of a similar length. The CRISPR/Cas systems are phylogenetically and functionally diverse, but each of these systems relies on three common steps: new sequence integration, CRISPR RNA biogenesis, and crRNA-guided target interference (Fig. 8.2c).

The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non­homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. CRISPRs are unevenly distributed between Bacteria and Archaea. Currently, CRISPR loci have been identified in 90 % of the archaeal genomes and 50 % of the bacterial genomes (Sorek et al. 2013). CRISPR-Cas systems have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish, human cells and plants (Belhaj et al. 2013; Gaj et al. 2013). In August 2012, Jinek et al. (2012) showed that a synthetic RNA chimera (single guide RNA, or sgRNA) created by fusing crRNA with tracrRNA is functional to a similar level as the crRNA and tracrRNA complex. As a result, the number of components in the CRISPR/Cas system was brought down to two, Cas9 and sgRNA (Jinek et al. 2012). For appli­cations in eukaryotic organisms, codon optimized versions of Cas9, which is orig­inally from the bacterium Streptococcus pyogenes, have been used. Four of the studies on the application of the CRISPR/Cas technology in plants used a plant codon-optimized version of Cas9, as using the previously described human codon — optimized version was not highly effective (Belhaj et al. 2013; Jiang et al. 2013).

All tested versions of Cas9 appear to work in plants with very high rates. Transgenic plants, generated using the CRISPR/Cas system, have been reported (up to 89 % for Arabidopsis and up to 92 % for rice) with bi-allelic mutation being recovered in the case of both plant species (Jiang et al. 2013). The discussed studies indicate the possibility of introducing functional CRISPR/Cas system in algae to target any sequence of choice, thus offering new opportunity for implementation in algal biotechnology for biomass production.

Together, these technologies promise to expand our ability to explore and alter any genome and constitute a new and promising paradigm to develop new synthetic biology tools for algal biofuels optimization.