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Guide RNA engineering for versatile Cas9 functionality - PubMed

  • ️Fri Jan 01 2016

Review

. 2016 Nov 16;44(20):9555-9564.

doi: 10.1093/nar/gkw908. Epub 2016 Oct 12.

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Review

Guide RNA engineering for versatile Cas9 functionality

Chance M Nowak et al. Nucleic Acids Res. 2016.

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats system allows a single guide RNA (sgRNA) to direct a protein with combined helicase and nuclease activity to the DNA. Streptococcus pyogenes Cas9 (SpCas9), a CRISPR-associated protein, has revolutionized our ability to probe and edit the human genome in vitro and in vivo Arguably, the true modularity of the Cas9 platform is conferred through the ease of sgRNA programmability as well as the degree of modifications the sgRNA can tolerate without compromising its association with SpCas9 and function. In this review, we focus on the properties and recent engineering advances of the sgRNA component in Cas9-mediated genome targeting.

© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.
Figure 1.

Streptococcus pyogenes CRISPR-SpCas9 guide RNA anatomy. (A) Endogenous CRISPR RNA (crRNA) and transacting crRNA (tracrRNA). The spacer sequence (orange) is 20 nucleotides in length and the repeat sequence (green) is 22 nucleotides that basepairs with tracrRNA complementary region (blue). The 3′ handle region (purple) has functional significance for structure-dependent recognition by SpCas9. (B) The synthetic sgRNA retains dual-tracrRNA:crRNA secondary structure via a fusion of the 3′ end of the crRNA to the 5′ end of the tracrRNA with an engineered tetraloop. (C) Individual functional modules of the sgRNA (sgRNA structure adopted from Briner et al., 2014). The 5′ spacer sequence dictates SpCas9 localization within the genome. The lower stem is formed by the duplex between the CRISPR repeat sequence from the crRNA and the region of complementarity in the tracrRNA. SpCas9 interacts with the upper and lower stems in a sequence-independent manner, whereas the bulge interactions with SpCas9 appear to be sequence-dependent. The nexus contains both sequence and structural features necessary for DNA cleavage and lies at the center of the sgRNA:SpCas9 interactions. The nexus also forms a junction between the sgRNA and both SpCas9 and the target DNA. The terminal hairpins assist in stabilizing the sgRNA and supports stable complex formation with SpCas9. The hairpins can also tolerate large deletion mutations and still exhibit cleavage activity.

Figure 2.
Figure 2.

sgRNA multiplexing strategies. (A) RNA endonuclease Csy4 recognizes a 28 nucleotide sequence flanking the sgRNA sequence and cleaves after the 20th nucleotide while remaining bound to the upstream region. This production strategy allows for RNAP II mediated transcription via a CMV promoter and polyadenylation signal. (B) The cis-acting ribozymes hammerhead ribozyme and HDV ribozyme flanking the 5′ and 3′ of the sgRNA, respectively, allow for self-cleaving production of sgRNAs and are not dependent on the presence of an exogenous protein. This production strategy also allows for RNAP II mediated transcription via a CMV promoter and polyadenylation signal. (C) Polycistronic tRNA–gRNA architecture allows the production of multiple sgRNAs from a single synthetic gene. Endogenous RNases RNaseP and RNase Z cleave the 5′ leader and 3′ trailer sequences at specific sites, respectively. This production strategy relies on the presence of an RNAP III promoter and terminator sequence, but achieves multiple sgRNA production via internal RNAP III promoter elements intrinsic to tRNA genes.

Figure 3.
Figure 3.

SpCas9 sgRNA mutational variants. (A) sgRNA variant in which the entire upper stem is removed and the bulge is replaced by a tetraloop that retains cleavage activity, suggesting that the upper stem may be dispensable. (B) sgRNA variant in which the spacer sequence is truncated from the canonical 20 nucleotides down to 14–15 nucleotides that allows catalytically active SpCas9 to still bind its target DNA without cleaving the target DNA. (C) sgRNA variant in which a putative RNAP III terminator sequence is removed from the lower stem by an A-U base pair flip and the upper stem is extended that increase sgRNA stability and enhance its assembly with SpCas9.

Figure 4.
Figure 4.

Incorporating RNA aptamer sequences into sgRNA. (A) MS2 loops that selectively bind MCP incorporated into the sgRNA 3′ end. (B) MS2 loops that selectively bind MCP incorporated into both the sgRNA tetraloop and the first hairpin. (C) MS2 loop incorporated in the 3′end in conjunction with a second aptamer hairpin f6 that has been selected to bind MCP. (D) CRISPRainbow sgRNA that utilizes three RNA binding protein-aptamer systems. The N22 peptide is fused to red fluorescent protein that binds the BoxB aptamer, the MCP peptide is fused to blue fluorescent protein and binds the MS2 aptamer and the PCP peptide fused to green fluorescent peptide binds the PP7 aptamer. Using different aptamers to bind red, green and blue fluorescent proteins the CRISPRainbow system creates seven different scaffolds that can be imaged as individual combinations of the primary colors. (E) sgRNA variant similar to the structure shown in Figure 4B, but also utilizes a truncated spacer sgRNA to achieve sgRNA multiplexing schemes that allow for both gene knockout and activation with catalytically active SpCas9.

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