The RNAi pathway has been studied in the unicellular green alga C. reinhardtii, and used as a reverse genetics tool in different algal species. Complex sets of endogenous small RNAs, including candidate microRNAs and small interfering RNAs, have been identified in four algal species, C. reinhardtii (Molnar et al. 2007; Zhao et al. 2007), Porphyra yezoensis (Liang et al. 2010), Phaeodactylum tricornutum (De Riso et al. 2009), and Ectocarpus siliculosus (Cock et al. 2010). However, RNAi mechanisms and their applications remain largely uncharacterized in most algae. RNAi against specific genes can be induced by the introduction of exogenously synthesized dsRNAs or siRNAs into cells or whole organisms (Cerutti et al. 2011; Moellering and Benning 2010; Molnar et al. 2009). Within algal species, in vitro — synthesized long dsRNAs have been electroporated into Euglena gracilis cells and shown to silence two endogenous genes homologous to the introduced dsRNAs (Iseki et al. 2002; Ishikawa et al. 2008). Recently, an RNAi triple knockdown of the three most abundant LHCII proteins (LHCBM1, 2 and 3) has been reported in Chlamydomonas with the aim of increasing the efficiency of photobiological H2 production (Oey et al. 2013). Artificial microRNA (amiRNA) expression success­fully exploits endogenous miRNA precursors to generate small RNAs that direct gene silencing in C. reinhardtii (Molnar et al. 2009; Schmollinger et al. 2010; Zhao et al. 2008). Zhao et al. (2009) developed an artificial amiRNA-based strategy to

knock down gene expression in Chlamydomonas using an endogenous Chlamydo — monas miRNA precursor (pre-miR1162) as the backbone. Other studies show that amiRNAs can be used as a highly specific, high-throughput silencing system, and they propose that they will become the system of choice for analysis of gene function in Chlamydomonas and related organisms (Molnar et al. 2009; Schmollinger et al.

2010) . The synthesized miRNAs provide a convenient tool for reverse genetic studies in Chlamydomonas. More recently, epitope-tagged protein-based amiRNA (ETPamir) screens were developed, in which target mRNAs encoding epitope — tagged proteins were constitutively or inducibly co-expressed in protoplasts with amiRNA candidates targeting single or multiple genes (Li et al. 2013). This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA

Fig. 8.2 Genome engineering tools. a miRNA pathway. MIR genes are transcribed by RNA polymerase II into pri-miRNA transcripts that are further processed into pre-miRNAs harboring a characteristic hairpin structure. From the stem of the pre-miRNA the miRNA/miRNA* duplex is excised by DCL1 and can be assisted by HYL and SE proteins. miRNA-guided AGO-containing RNA-induced silencing complex (RISC) directs mRNA cleavage or translation inhibition of the target transcript. b Summary of Transcription activator-like effectors (TALEs) nuclease. Custom — designed nucleases introduce double-strand breaks with high precision at predetermined genomic loci. Double-strand breaks are either repaired by error-prone non-homologous end-joining (NHEJ) or high fidelity homologous recombination (HR). NHEJ repair causes random insertions and/or deletions of nucleotides around the target site and some of these mutations will knockout gene function. Gene replacement, tagging, or correction can be achieved by HR-mediated targeted integration of a donor construct that is provided together with a nuclease pair. c CRISPR/Cas9 mediated target DNA cleavage. The CRISPR loci include Cas genes, a leader sequence, and several spacer sequences derived from engineered or foreign DNA that are separated by short direct repeat sequences. Cleavage occurs on both strands, 3 bp upstream of the NGG proto-spacer adjacent motif (PAM) sequence on the 3′ end of the target sequence and is followed by DNA repair by the endogenous cellular repair machinery expression/processing and antibody unavailability. These screens could improve algal biofuel engineering research by making amiRNA a more predictable and manageable genetic and functional genomic technology. From a practical perspec­tive, RNAi is becoming a customary method for directed gene silencing in algae. As the necessary molecular tools are developed, RNAi approaches are expected to contribute to the functional characterization of novel genes, as well as to the strain engineering of algae (Fig. 8.2a). Ultimately, RNAi technology may provide much-needed insights into gene function, metabolic pathways, and regulatory networks allowing us to comprehend the role of algal species in nature, as well as to engineer these organisms for the synthesis of valuable bioproducts.