PerspectiveRNA Events

Cas9 Targeting and the CRISPR Revolution

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Science  16 May 2014:
Vol. 344, Issue 6185, pp. 707-708
DOI: 10.1126/science.1252964

The ability to add, remove, or change DNA sequences is essential to studies that investigate the genetic underpinning of phenotypic traits. With its unprecedented efficiency and stunning ease of use, DNA editing technology based on the prokaryotic CRISPR (clustered regularly interspersed short palindromic repeats)–Cas9 system is completely revolutionizing genome engineering. In little more than a year, CRISPR-Cas9 editing has been implemented in a multitude of model organisms and cell types (1) and has already started to supplant incumbent genome editing technologies, such as TALENs (transcription activator-like effector nucleases) and ZFNs (zinc finger nucleases).

The CRISPR-Cas9 editing technology is derived from the CRISPR adaptive immune system of bacteria (2) and consists of an RNA-guided endonuclease (Cas9 being one example) harnessed for specific targeting and cleavage of DNA by means of a single programmable RNA molecule (3). Following precise DNA cleavage of the sequence of interest, mutations are surgically inserted using errorprone DNA repair mechanisms or designed templates, generating altered genotypes (46). To further harness this versatile technology platform for scalable genome editing, transcriptional control, DNA labeling, and so forth, as well as to minimize possible off-target effects, a more precise molecular understanding of how Cas9 targets and interacts with DNA is required. Recent work by Sternberg et al. (7) revealed the molecular mechanisms by which the endonuclease Cas9 interrogates DNA, providing a basis for improving the specificity and efficiency of CRISPR technologies for genome editing and beyond.

In the CRISPR-Cas9 adaptive immune system, the Cas9 endonuclease is guided by an RNA duplex (8), including the CRISPR RNA (crRNA), which directs sequence-specific double-stranded DNA (dsDNA) cleavage (9). After R-loop formation, where the crRNA displaces one of the DNA strands and forms a short DNA-RNA segment with the other DNA strand, the HNH nuclease domain of Cas9 nicks the DNA strand complementary to the guide RNA, while the RuvC domain cleaves the displaced DNA strand, yielding a double-strand break (3, 10) (see the figure). Specificity is determined by the crRNA sequence, which targets complementary DNA, flanked by a short proto-spacer adjacent motif (PAM). A single guide RNA (sgRNA) molecule can be engineered to replace the native RNA duplex (3), and the resulting sgRNA-Cas9 system can be readily deployed for sequence-specific DNA cleavage of virtually any sequence of interest (46), because the three-nucleotide NGG PAM consensus associated with the widely used Streptococcus pyogenes Cas9 (SpyCas9) is present at high frequency in random DNA sequences. Yet how Cas9 specifically and efficiently localizes a single DNA target in the vast maze of a genome has remained enigmatic.

Targeting a molecular scalpel.

The CRISPR-Cas9 nuclease, programmed with sgRNA, specifically targets a short DNA sequence “tag” (the PAM) and unzips DNA complementary to the sgRNA to create a sgRNA–target DNA heteroduplex, triggering R-loop formation. Two nuclease domains (RuvC, HNH) each nick one DNA strand, generating a double-strand break. Structurally, the Cas9 recognition (REC) domain interacts with the sgRNA, while the nuclease (NUC) lobe drives interaction with the PAM and target DNA.

Using single-molecule and bulk biochemical experiments, Sternberg et al. have unraveled the molecular mechanisms by which Cas9 programmed with sgRNA scans DNA molecules, localizes the cleavage target, and specifically interacts with PAM sequences. Using tethered “curtains” of single DNA molecules together with fluorescence microscopy, they visualized labeled Cas9 interacting with phage DNA. Cas9 programmed with sgRNA complementary to various sites in the tethered DNA localized almost exclusively at the predicted target sites. Facilitated diffusion by sliding along the DNA was not important for specific Cas9-DNA interactions; rather, targets were identified through three-dimensional collisions between Cas9 and DNA, with PAM distribution and density playing a critical role in both Cas9 localization and binding lifetimes. PAM binding by Cas9 is long-lived in the presence of the target sequence, whereas the interaction is transient with rapid dissociation in the absence of target sequences. The critical role of the PAM in initiating the interaction between DNA and Cas9 is best illustrated by the absence of binding to substrates that contain the target sequence but lack a flanking PAM. This is consistent with short-lived Cas9 interactions with molecules that have high PAM densities but lack sgRNA complementarity.

Once DNA interrogation is initiated by Cas9 at the PAM site, the RNA guide is unzipped and inserted into the DNA duplex to directionally unwind the complementary dsDNA double helix toward the distal end of the target sequence, creating a sgRNA–target DNA heteroduplex and triggering R-loop formation. Following target dsDNA destabilization at the PAM, a stretch of several complementary bases between the sgRNA and the target DNA is critical to ensure sustainable unwinding and heteroduplex extension. Once established, this interaction catalytically activates the two nickase domains of the endonuclease to specifically generate a double-strand break in the target DNA molecule, exactly three nucleotides into the sequence homologous to the guide.

Until recently, the crRNA sequence was thought to be all-important for Cas9 targeting of complementary DNA. Sternberg et al. have now established that PAMs are the key drivers of target DNA interrogation, helping to initiate the interaction, and that the sgRNA subsequently directs cleavage only of complementary DNA sequences. Results indicate that Cas9 remains bound to both ends of the cleaved DNA molecules, perhaps extending the lifetime of cleaved termini and allowing recruitment of the DNA repair machinery.

Recent structural studies have shed light on the detailed molecular interactions between Cas9 and the sgRNA, as well as Cas9 and the target DNA (11, 12). Cas9 has a distinct bilobed architecture, with a sgRNA-binding target recognition lobe and a nuclease domain, which is hypothesized to carry the PAM interaction domain. The sgRNA–target DNA heteroduplex is formed in the groove located between these two lobes. The structural understanding of these interactions will facilitate structure-based rational design of optimal guides by defining the elements that drive PAM specificity, binding fidelity, and mismatch tolerance for various Cas9 protein families, as well as the similarities and differences between different CRISPR systems.

An intriguing question is whether there are nucleotide preferences within PAM sequences that could optimize Cas9 efficiency and/or specificity and thereby minimize potential off-target cleavage. This problem has already been partially addressed by using multiplexing, dual nicking guides, and short sgRNAs (13). The study by Sternberg et al. indicates that off-target concerns should be exclusively limited to sites homologous to sgRNA sequences that are flanked by a PAM.

Major factors underlying the CRISPRCas9 genome editing revolution include the compactness, simplicity, and targeting flexibility afforded by this system (13). The implications for enhanced Cas9 targeting specificity and binding efficiency are exciting and establish a basis for the further optimization of the Cas9-sgRNA system and the development of next-generation CRISPR tools. Future studies will determine the potential of various natural or engineered Cas9 proteins, sgRNA molecules, and their respective PAM sequences for increased sequence recognition specificity and DNA binding efficiency, and possibly the generation of short Cas9 homologs for convenient packaging and delivery. Notwithstanding the CRISPR craze of 2013 (1), this dynamic field is off to an effervescent start for 2014, and these latest findings open new engineering avenues for CRISPR-Cas9 and set the stage for further applications in synthetic biology, translational research, and next-generation genome engineering.

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