SEE ALSO CRISPR-Cas

Background

Highlights

• CRISPR-Cas9 is a powerful tool to modulate transcription in wide range of cell types.
• An expanding set of CRISPR-based transcription effectors is available.
• Gene networks can be efficiently probed and modified for biotechnology applications.^[1]

CRISPR-Cas9 has recently emerged as a promising system for multiplexed genome editing as well as epigenome and transcriptome perturbation. Due to its specificity, ease of use and highly modular programmable nature, it has been widely adopted for a variety of applications such as genome editing, transcriptional inhibition and activation, genetic screening, DNA localization imaging, and many more. In this review, we will discuss non-editing applications of CRISPR-Cas9 for transcriptome perturbation, metabolic engineering, and synthetic biology.^[1]

Since the early days of genetic engineering there has been a need for control of gene expression. Naturally occurring transcription factors (TFs) have traditionally been used to achieve this goal (reviewed in ^[2]). However, their limited DNA binding sequence space required installing specific sequences within the transcription regulatory elements of the target genes. This can be technically difficult and may have unintended consequences on gene expression. Zinc fingers (ZFs) and transcription activator-like effectors (TALEs) were developed to overcome the fixed binding sequence requirements of native TFs. However, both ZFs and TALEs have significant limitations. ZFs have complicated design criteria and large highly repetitive TALE genes are difficult to synthesize and clone (reviewed in ^[3][4]). These challenges have recently been overcome using CRISPR-Cas9 based TFs. The biochemical properties of CRISPR-Cas9 based TFs that enable such flexibility and describe their applications to synthetic gene circuit design and multi-plexed perturbation of native gene networks.^[1]

Transcriptional regulation with CRISPR-Cas9

Figure 1. Overview of Cas9 nuclease and dCas9-based transcription factors. (a) Wild-type Cas9 endonuclease guided by crRNA:tracrRNA to a specific site in DNA creates a double-stranded DNA break. (b) dCas9, nuclease deactivated mutant of Cas9, is an RNA programmable DNA binding protein. It can act as a steric repressor of transcription in prokaryotes and eukaryotes. sgRNA is an artificial chimeric molecule consisting of crRNA and tracrRNA molecules connected with a short loop. (c) dCas9 fusion with various transcription effectors can be used to repress or activate transcription. (d) Effector domains can be recruited by sgRNA in addition to dCas9 for enhanced activity. (e) sgRNA can be modified with specific protein binding hairpins to concurrently recruit repressor or activator domains in the same cell.^[1]

Cas9 is a key protein of bacterial Type II CRISPR adaptive immune system (reviewed in ^[5]). In its native context, Cas9 is an RNA-guided endonuclease that is responsible for targeted degradation of the invading foreign DNA–plasmids and phages. Cas9 is directed to its DNA targets by forming a ribonucleoprotein complex with two small non-coding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (Figure 1a). In the less common class 2 CRISPR-Cas systems (types II, V, and VI), which are almost completely restricted to bacteria, the effector complex is represented by a single multidomain protein ^[6]. The best-characterized class 2 effector is Cas9 (type II), the RNA-dependent endonuclease that contains two unrelated nuclease domains, HNH and RuvC, that are responsible for the cleavage of the target and the displaced strand, respectively, in the crRNA–target DNA complex (, 4zt0). The type II loci also encode a trans-acting CRISPR RNA (tracrRNA) that evolved from the corresponding CRISPR repeat and is essential for pre-crRNA processing and target recognition in type II systems. Cas9 is directed to its DNA targets by forming a ribonucleoprotein complex with these 2 small non-coding RNAs: crRNA and tracrRNA. By elegant engineering, (4zt9^[7]) that too efficiently directs Cas9 protein to DNA targets encoded within the guide sequence of sgRNA ^[8]:

Examples of 3D structures of single guide RNA (sgRNA)

• (5fw2).
• (5b2s).
• (5b2p).
• (4axw).

The , termed the guide sequence, adjacent to a ^[8][9]. Despite this, a ^{[8][10][11][12]}, more so within the 5’ proximal position of the guide sequence.

• ^[7] (4cmp^[13]).
• (4zt0)^[7].
• (4oo8)^[14].
• .

• (5f9r)^[15].
• .
• - place of accurate, precise, and programmable DNA cleavage.
• (sgRNA is not shown).

In the type II-A system, the Cas9-tracrRNA complex and Csn2 are involved in spacer acquisition along with the Cas1-Cas2 complex ^[16][17]; the involvement of Cas9 in adaptation is likely to be a general feature of type II systems. Although the key residues of Cas9 involved in PAM recognition are dispensable for spacer acquisition, they are essential for the incorporation of new spacers with the correct PAM sequence ^[17]. The involvement of Cas9 in PAM recognition and protospacer selection ^[17] suggests that in type II systems Cas1 may have lost this role.

Cas9 nuclease can be converted into (PDB entry 4zt9), an RNA-programmable DNA-binding protein, by (Figure 1b) ^[7][1][8][9].

In the simplest case, dCas9 can repress transcription by sterically interfering with transcription initiation or elongationby being targeted to the gene of interest with a properly chosen sgRNA ^{[8][9][10][11][18][19][20][21][22]}. The repression strength is strongly dependent on the position with respect to the target promoter as well as the nature of promoter itself ^{[10][11][18][19]}. In prokaryotes, repression of up to 1000-fold was achieved when targeting dCas9 to either DNA strand within a promoter or to the non-template DNA strand downstream. However, in eukaryotic cells such steric repression is weaker: only up to 2-fold and 20-fold repression was observed with natural promoters in mammalian and yeast cells correspondingly^[10][20][21]. As a notable exception, synthetic promoters specifically constructed for direct repression by dCas9 can be repressed up to 100-fold in mammalian cells^[22].

Cas9-sgRNA-target DNA complexes from Streptococcus pyogenes:

• , 4zt0)
• (5fw2).
• (5b2s).
• (4oo8).
• (5f9r).

Other representatives: 5y36, 4un3.

Crystal Structure of Staphylococcus aureus Cas9^[23]

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer adjacent motif (PAM) and complementarity to the guide RNA. Recently, we harnessed Staphylococcus aureus Cas9 (SaCas9), which is significantly smaller than Streptococcus pyogenes Cas9 (SpCas9), to facilitate efficient in vivo genome editing. Here, the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double stranded DNA targets, containing the 5'-TTGAAT-3' PAM and the 5'-TTGGGT-3' PAM, at 2.6 and 2.7 A˚ resolutions, respectively, were reported. The structures revealed the mechanism of the relaxed recognition of the 5'-NNGRRT-3' PAM by SaCas9. A structural comparison of SaCas9 with SpCas9 highlighted both structural conservation and divergence, explaining their distinct PAM specificities and orthologous sgRNA recognition.

Overall Structure of the SaCas9–sgRNA–Target DNA Complex

The (residues 1–1053; N580A/C946A) in complex with a 73-nucleotide (nt) sgRNA, a 28-nt target DNA strand and an 8-nt non-target DNA strand, containing the 5'-TTGAAT-3' PAM was solved (5czz). SaCas9 adopts a consisting of a REC lobe (residues 41–425) and a NUC lobe (residues 1–40 and 435–1053). The by an arginine-rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The the RuvC (residues 1–40, 435–480 and 650–774), HNH (residues 520–628), WED (residues 788–909), and PI (residues 910–1053) domains. The can be divided into a Topoisomerase-homology (TOPO) domain and a C-terminal domain (CTD). The three separate motifs (RuvC-I–III) and interacts with the HNH and PI domains. The is connected to RuvC-II and RuvC-III by the L1 (residues 481–519) and L2 (residues 629–649) linker regions, respectively. The active site of the HNH domain is distant from the cleavage site in the target DNA strand (the phosphodiester linkage between dC3 and dA4), indicating that the present structure represents the inactive state. The WED and RuvC domains are connected by a loop (residues 775–787). Previous structural studies revealed that SpCas9 undergoes conformational rearrangements upon guide RNA binding, to form the central channel between the REC and NUC lobes. In the absence of the guide RNA, SpCas9 and AnCas9 adopt a closed conformation, where the active site of the HNH domain is covered by the RuvC domain. In contrast, the ternary and quaternary complex structures of SpCas9 adopt an open conformation and have the central channel, which accommodates the guide RNA–target DNA heteroduplex (referred to as the guide:target heteroduplex). The present , suggesting that the guide RNA-induced conformational

activation is conserved between SaCas9 and SpCas9. A structural comparison between SaCas9 and SpCas9 revealed that, although their overall architectures are similar, there are notable differences in their REC, WED, and PI domains, as described in detail below, thereby explaining the significant sequence and size differences of the two Cas9 orthologs.

Structure of the sgRNA–Target DNA Complex

The SaCas9 sgRNA consists of the guide region (G1–C20),repeat region (G21–G34), tetraloop (G35–A38), anti-repeat region (C39–C54), stem loop 1 (A56–G68), and single-stranded linker (U69–U73), with A55 connecting the anti-repeat region and stem loop 1. U73 at the 3' end is disordered in the present structure. The guide region (G1–C20) and the target DNA strand (dG1–dC20) form the , whereas the target DNA strand (dC(8)–dA(1)) and the non-target DNA strand (dT1*–dG8*) form a (referred to as the PAM duplex). The repeat (G21–G34) and anti-repeat (C39–C54) regions form a distorted duplex (referred to as the ) via 13 Watson-Crick base pairs. is formed via three Watson-Crick base pairs (G57:C67–C59:G65) and two non-canonical base pairs (A56:G68 and A60:A63). U64 does not base pair with A60 and is flipped out of the stem loop. The N1 and N6 of A63 hydrogen bond with the 2'-OH and N3 of A60, respectively. G68 stacks with G57:C67, with the G68 N2 interacting with the backbone phosphate group between A55 and A56. A55 adopts the syn conformation, and its adenine base stacks with U69. In addition, the N1 of A55 hydrogen bonds with the 2'-OH of G68, thus stabilizing the basal region of stem loop 1. An adenosine residue immediately after the repeat:anti-repeat duplex is highly conserved among CRISPR-Cas9 systems, and the equivalent adenosine in the SpCas9 sgRNA, A51, also adopts the syn conformation, suggesting that these adenosine residues play conserved key roles in connecting the repeat:anti-repeat duplex and stem loop 1.

Recognition Mechanism of the Guide:Target Heteroduplex

The formed between the REC and NUC lobes. The sugar-phosphate backbone of the PAM-distal region (A3–U6) of the sgRNA interacts with the . In SpCas9 and SaCas9, the RNA–DNA base pairing in the 8 bp PAM-proximal ‘‘seed’’ region in the guide:target heteroduplex is critical for Cas9-catalyzed DNA cleavage. Consistent with this, the phosphate backbone of the sgRNA seed region (C13–C20) is extensively recognized , as in the case of SpCas9. These structural observations explain the RNA-guided DNA targeting mechanism of SaCas9. The C-terminal region of the REC lobe interacts with the PAM-distal region of the heteroduplex, whereas the N-terminal region of the REC lobe interacts with the repeat:anti-repeat duplex and the PAM-proximal region of the heteroduplex. Notably, the C-terminal region of the REC lobe of SaCas9 shares structural similarity with those of SpCas9 (PDB: 4un3, 26% identity, rmsd of 1.9 A˚ for 177 equivalent Ca atoms) and AnCas9 (PDB: 4oge, 16% identity, rmsd of 3.2 A˚ for 167 equivalent Ca atoms). These structural findings suggested that the Cas9 orthologs recognize the PAM-distal region of the guide:target heteroduplex in a similar manner.

Recognition Mechanism of the sgRNA Scaffold

The . Consistent with our data showing that the distorted repeat:anti-repeat duplex is critical for Cas9-catalyzed DNA cleavage, the . The 2'-OH of C30 hydrogen bonds with , and the backbone phosphate groups of U31, C45, and U46 interact with , respectively. These structural observations explain the structure-dependent recognition of the repeat:anti-repeat duplex by SaCas9. Stem loop 1 is recognized by the bridge helix and the REC lobe. The phosphate backbone of interacts with the bridge helix () and the REC lobe (). The . The flipped-out , respectively. A55 is extensively recognized by the phosphate lock loop. The N6, N7, and 2'-OH of A55 hydrogen bond with Asn780/Arg781, Leu783, and Lys906, respectively. Lys57 interacts with the backbone phosphate group between C54 and A55, and the side chain of Leu783 forms hydrophobic contacts with the nucleobases of A55 and A56. The phosphate backbone of the linker region electrostatically interacts with the RuvC domain (Arg452, Lys459, and Arg774) and the phosphate lock loop (Arg781), and the nucleobase of G70 stacks with the side chain of Arg47 on the bridge helix.

Structural Basis for the Orthogonal Recognition of sgRNA Scaffolds

A comparison of the quaternary complex structures of SaCas9 and SpCas9 revealed that the structurally diverse REC and WED domains recognize distinct structural features of the repeat:anti-repeat duplex, allowing the cognate sgRNAs to be distinguished in a highly specific manner. The SaCas9 WED domain has a new fold comprising a twisted five-stranded β-sheet flanked by four α-helices, and is responsible for the recognition of the distorted repeat:anti-repeat duplex, as described above. In contrast, the SpCas9 WED domain adopts a compact loop conformation and interacts with the repeat:anti-repeat duplex, which is structurally different from that of the SaCas9 sgRNA. The AnCas9 WED domain has yet another different fold containing three antiparallel b-hairpins. These structural differences in the WED domains are consistent with the variations in the sgRNA scaffolds among the CRISPR-Cas9 systems. The REC lobes also contribute to the orthogonal recognition of the sgRNA scaffolds. While the REC lobes of SaCas9 and SpCas9 share structural similarity, the SpCas9 REC lobe has four characteristic insertions (Ins 1–4), which are absent in the SaCas9 REC lobe. Ins 2 (also known as the REC2 domain) does not contact the nucleic acids in the SpCas9 structures and is dispensable for the DNA cleavage activity, consistent with the absence of Ins 2 in SaCas9 (Figures 4E and 4F). Ins 1 and 3 recognize the SpCas9-specific internal loop in the repeat:anti-repeat duplex (Figure 4F and Figure S6C). In particular, Ins 3 interacts with the flipped-out G43 and U44 in the repeat:anti-repeat duplex in base-specific manners (Figure S6C). In addition, Ins 4 interacts with stem loop 1 of the SpCas9 sgRNA, which is shorter than that of the SaCas9 sgRNA. Together, these structural observations demonstrate how the Cas9 orthologs recognize their cognate sgRNAs in orthogonal manners, using specific combinations of the structurally diverse REC and WED domains.

See aslo

• Cas9
• Endonuclease