Advances in Industrial Biotechnology Using CRISPR-Cas Systems Paul D. Donohoue, Rodolphe Barrangou, Andrew P. May  Trends in Biotechnology  Volume 36, Issue 2, Pages 134-146 (February 2018) DOI: 10.1016/j.tibtech.2017.07.007 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated (CRISPR-Cas) Adaptive Immunity. The three stages of CRISPR-Cas adaptive immunity are adaptation, expression, and interference. In the adaptation phase (A), the host is invaded by foreign DNA during infection. (B) Various cas genes are expressed and bind portions of invading DNA (protospacer). Once bound, (C) the DNA sequence is then incorporated adjacent to the leader sequence of the CRISPR array, flanked by a repeat sequence (gray diamonds). Upon subsequent infection, the expression phase (D) is initiated by transcription of the CRISPR array as a single transcript, and (E) individual spacer-repeat elements are processed by an endonuclease into CRISPR RNA (crRNA). (F) The crRNA is subsequently bound by the Cas nuclease. The Cas:crRNA complex is then able to (G) probe invading DNA for a complementary protospacer sequence. Interference occurs when (H) the Cas:crRNA complex identifies a sequence that is complementary to the spacer and adjacent to the protospacer adjacent motif (PAM). (I) The invading DNA is cleaved by Cas-nuclease to prevent infection. Trends in Biotechnology 2018 36, 134-146DOI: (10.1016/j.tibtech.2017.07.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Array Genotyping. Each colored rectangle represents a unique spacer; absent spacers and repeats are represented by an ‘X’; gray diamonds represent CRISPR repeats. New spacers are incorporated adjacent to the CRISPR leader sequences (black arrow), while older spacers reside distal to the leader sequence as the array grows through the introduction of new spacers. (A) Isolated strains that contain an identical collection of spacers. (B) Closely related strains with iterative acquisition of new spacers. (C) Isolates that have a mixed diversity of acquired spacers show deviation from an ancestrally similar collection of spacers. (D) Strains having no shared spacers between them. Trends in Biotechnology 2018 36, 134-146DOI: (10.1016/j.tibtech.2017.07.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Key Figure: Modes of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated (CRISPR-Cas) Targeting Cas9 DNA Cleavage (A) Schematic of a Cas9 protein and single guide (sg)RNA complex cleaving a double-stranded (ds)DNA target. (B) Streptococcus pyogenes sgRNA and Cas9 protein in complex with a dsDNA target. The spacer sequence hybridizes to the target strand of the targeted DNA and the Cas9 nuclease generates a DS break (DSB) (red arrows) three nucleotides upstream of the 5′-NGG-3′ protospacer adjacent motif (PAM) sequence. After the Cas9:sgRNA disassociates from the target sequence, (C) the DSB is repaired through endogenous DNA repair pathways, either engaging the nonhomologous end joining (NHEJ) repair pathway, resulting in resection (red arrows) or insertion (not depicted) of nucleotides at the DSB, followed by rejoining of the DNA strands and the introduction of insertions and/or deletions (indels) to the sequence. Alternatively, the (D) homology-directed repair (HDR) pathway is initiated by the presence of a donor template with homology to the break site, resulting in a crossover event (broken black lines). The break is repaired by incorporation of the exogenous sequence into the genomic DNA. Transcriptional repression. (E) Nuclease deactivated Cas9 (dCas9) is targeted to bind upstream of an open reading frame (ORF), which blocks binding of RNA polymerase (RNAP) and prevents gene transcription. CRISPR interference/activation (CRISPRi/a) utilizes a dCas9:sgRNA complex and a transcriptional regulatory domain, which is species and application specific. The regulatory domain may be recruited to the Cas nuclease (F) by fusing an RNA-binding domain to the regulatory domain and adding an RNA hairpin to the 3′ end of the sgRNA, depicted here as resulting in the upregulation of transcription; alternatively, (G) the regulatory domain may be directly fused to the dCas9, modifying the DNA target region and preventing RNAP binding and gene transcription. The use of guide modification or dCas9 protein fusion is suitable in both CRISPRi and CRISPRa applications. Trends in Biotechnology 2018 36, 134-146DOI: (10.1016/j.tibtech.2017.07.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Biological Circuits. (A) Schematic representation of a synthetic CRISPR-mediated gene circuit to control the transcription of a reporter gene (repT). Deactivated Cas9 (dCas9) is put under the control of an inducible promoter, here represented by the promoter pBAD, which is activated when cells are provided with arabinose (Ara). A single guide (sg)RNA, directed to target the transcriptional start site (TSS) of repT, is constitutively expressed by a Type III RNA promoter (e.g., T7 or U6). Introduction of Ara results in the inductions of dCas9 expression, which subsequently binds the transcribed sgRNA and targets the repT TSS, causing a repression of transcription. (B) A complex gene circuit wherein the sgRNA is incorporated into the 3′ region of the open reading frame of enz-B, whose expression is under the control of a lac operon (lacO), which is inducible in the presence of the lactose-analog IPTG. The sgRNA is flanked by two ribozymes (riboZ), allowing for excision of the sgRNA before enz-B translation. The sgRNA targets the TSS of enz-A, which mediates the reduction of phenylacetic into phenylacetaldehyde. enz-B encodes a protein in a competing pathway, where the shared precursor, phenylacetic, is decarboxylated to yield the desired endproduct, toluene. Introduction of Ara and IPTG drives the expression of dCas9 and the enz-B ORF. dCas9 binds the ribozyme-liberated sgRNA, and inhibits enz-A expression, while enz-B catalyzes the conversion of phenylacetic to toluene. Adapted from [80]. Trends in Biotechnology 2018 36, 134-146DOI: (10.1016/j.tibtech.2017.07.007) Copyright © 2017 Elsevier Ltd Terms and Conditions