Outlook

CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats) evolved in bacteria and archaea, and provide for adaptive immunity against foreign DNA, such as that from invading viruses. Modified CRISPR-Cas systems have dramatically transformed our ability to edit the genomes of different organisms, and paved the way toward targeted therapeutic interventions, most notably the prospect of targeted genome editing to correct genetic disorders and possibly to disrupt invading viral genomes. I believe that tools based on Cas9, a unifying factor capable of colocalizing RNA, DNA and protein will confer unprecedented control over cellular organization, regulation and behavior. Here I will review CRISPR-Cas targeting methodology, prospective engineering advances, and suggest potential applications ranging from basic science to the clinic.

Origin of CRISPR-Cas Systems

Bacteria and archaea have evolved adaptive immune defenses to protect themselves against foreign genetic material. They are termed CRISPR-CRISPR-associated-systems (CRISPR-Cas) and use short RNA to direct degradation of foreign nucleic acids (1-3). CRISPR-Cas systems work by incorporating fragments of invading phage or plasmid DNA into CRISPR loci (the immunization phase), and using the corresponding transcribed CRISPR RNAs (crRNAs) to guide the degradation of homologous sequences (4). Each CRISPR locus encodes acquired ‘spacers’ (fragments of foreign DNA) that are separated by repeat sequences. Transcription of such a locus yields a pre-crRNA, which is processed to generate crRNAs consisting of spacer-repeat fragments that guide effector nuclease complexes to cleave dsDNA sequences complementary to the spacer. Hence, expressing or delivering different crRNAs will readily retarget the CRISPR system toward different dsDNA sequences (5-7).

Engineering CRISPR-Cas Systems

Cas9 effector nuclease is an RNA-guided dsDNA-binding protein and the first identified programmable unifying factor capable of colocalizing all three types of sequence-defined biological polymers, making it an invaluable tool for engineering living systems. An important step towards effective genome editing and genome regulation is the development of type II CRISPR-Cas systems that enable robust RNA-guided genome modifications in multiple eukaryotic systems (8-10). The type II effector system is comprised of a long pre-crRNA transcribed from the spacer-repeat CRSPR locus, the multifunctional Cas9 protein and a trans-activating crRNA (tracrRNA) important for processing the pre-crRNA and formation of the Cas9 complex.

Type II CRISPR interference is a multistep process (11). First, tracrRNAs hybridize to repeat regions of the pre-crRNA. Second, endogenous RNase III cleaves the hybridized crRNA-tracrRNAs, and a second event removes the 5′ end of each spacer, yielding mature crRNAs that remain associated with both the tracrRNA and Cas9. Third, each mature complex locates a target dsDNA sequence and cuts both strands. To recognize and cleave its target, crRNA-tracrRNA-Cas9 requires both sequence complementarity between the spacer and the target ‘protospacer’ sequence as well as the presence of an appropriate protospacer adjacent motif (PAM) sequence at the 3’ end of the protospacer sequence (12). The PAM is an essential targeting component that also serves as a self versus non-self recognition system to prevent the CRISPR locus itself from being targeted. Different PAM requirements for different type II system should also be considered when designing a CRISPR targeting system. The most commonly engineered system thus far, that of Streptococcus pyogenes, requires a PAM with sequence NGG, where N is any nucleotide. Type II systems may differ in the details of pre-crRNA production and crRNA-tracrRNA processing (13).

Implementing CRISPR-Cas technology in a given organism requires the efficient reconstitution of the crRNA-tracrRNA-Cas9 functional unit. Since bacteria are natively using CRISPR, the system can be used as it is. Using CRSPR technology in humans requires, however, a few adjustments. These involve expression of a human-codon-optimized Cas9 protein with an appropriate nuclear localization signal, and the crRNA and tracrRNA expressed either individually or as a single chimera via a RNA polymerase III promoter (14,15). The most common approach is to express a chimeric crRNA-tracrRNA, also termed a short guide RNA (sgRNA), providing for enhanced simplicity and robust targeting, especially if the sgRNA is not truncated (16). This methodology has already been used to edit the genomes of numerous model eukaryotic organisms.

Toward Cas9 Therapeutics

Cas9-mediated therapeutic interventions may take two paths. The first entails targeted genome editing to correct genetic disorders (17-19) and possibly to disrupt invading viral genomes. The second will use Cas9nuclease-null fusions for targeted genome regulation in a manner similar to the use of small-molecule drugs, with the advantage that both repression and activation modalities would be available. One could potentially intervene using such an approach to correct epigenetic misregulation of gene expression, to control inflammation and autoimmunity, and also to repress transcription of viral genes or even viral co-receptors in vulnerable cell types.

Therapeutic implementation of Cas9 systems will require first resolving several technical difficulties. First, Cas9-encoding cassettes must be efficiently delivered to target cells in vivo. Unfortunately, Cas9 proteins are quite large; the commonly used Cas9 from S. pyogenes is 1,368 amino acids in length. Strategies to reduce the size of the protein could include employing smaller Cas9 orthologs or engineering a minimal Cas9 by removing domains not essential for the intended functionality. The Cas9 protein could then be efficiently packaged into viral vectors (adeno-associated viruses, adenoviral vectors and lentiviruses) for direct in vivo delivery (20-22). The use of tightly regulated expression vectors together with the ability to enable both transient and controlled release of targeting reagents will be critical to restricting the resulting Cas9-mediated functions to specific tissues.

Apart from improving gene delivery, work must be done to improve the specificity of Cas9 binding. Resolving this obstacle is of the utmost importance, as even highly specific methodologies, when applied to sufficient numbers of cells, lead to off-target activity that results in a risk of oncogenesis. Prospective approaches would require cooperativity through offset nicking and biasing repair outcomes toward HR (homologous recombination) versus NHEJ (nonhomologous end joining). Also highly useful would be colocalizing target and donor DNA via direct guide RNA tethering or Cas9 recombinases and transposases, especially in targeting post-mitotic cells where the function of the endogenous HR machinery is usually diminished.

Avoiding an adverse immune response is also critical. The classical immune suppression may be useful for the duration of treatment, but less so for long-term regulatory modifications. A more promising approach would be to ‘humanise’ the relevant peptide fragments responsible for Cas9 immunogenicity, as has been accomplished for antibody therapeutics (23). Finally, the functional flexibility of Cas9 may permit the mimicry of strategies used by viruses such as disrupting the major histocompatibility complex trafficking machinery (24).

Additional synergistic technologies will be needed to fully realise Cas9’s therapeutic potential, most notably efficient, targeted and safe in vivo gene-delivery vehicles. If ex vivo genome targeting proves effective (for instance, in hematopoietic stem cells), then the ability to rapidly retrieve and screen modified cells will be critical. In conclusion, the versatility and ease of use offered by Cas9, together with its singular ability to bring together RNA, DNA, and protein in a fully programmable fashion will offer the support to develop a powerful toolset for the regulation and monitoring of complex biological systems.

Functioning of the type II CRISPR-Cas systems in bacteria. Phase 1: in the immunisation phase, the CRISPR system stores the molecular signature of a previous infection by integrating fragments of invading phage or plasmid DNA into the CRISPR locus as ‘spacers’. Phase 2: in the immunity phase, the bacterium uses this stored information to defend against invading pathogens by transcribing the locus and processing the resulting transcript to produce CRISPR RNAs (crRNAs) that guide effector nucleases to locate and cleave nucleic acids complementary to the spacer.

Author: Sebastian Florescu, PhD

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