Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

σ1 receptor ligands control a switch between passive and active threat responses.

Humans and many animals show 'freezing' behavior in response to threatening stimuli. In humans, inappropriate threat responses are fundamental characteristics of several mental illnesses. To identify small molecules that modulate threat responses, we developed a high-throughput behavioral assay in zebrafish (Danio rerio) and evaluated 10,000 compounds for their effects on freezing behavior. We found three classes of compounds that switch the threat response from freezing to escape-like behavior. We then screened these for binding activity across 45 candidate targets. Using target profile clustering, we identified the sigma-1 (σ1) receptor as having a role in the mechanism of behavioral switching and confirmed that known σ1 ligands also disrupt freezing behavior. Furthermore, mutation of the gene encoding σ1 prevented the behavioral effect of escape-inducing compounds. One compound, which we call finazine, potently bound mammalian σ1 and altered threat-response behavior in mice. Thus, pharmacological and genetic interrogation of the freezing response revealed σ1 as a mediator of threat responses in vertebrates.

Targeted Mutagenesis in Zebrafish Using CRISPR RNA-Guided Nucleases.

In recent years, the zebrafish has become a critical contributor to various areas of biomedical research, advancing our fundamental understanding of biomedicine and helping discover candidate therapeutics for human diseases. Nevertheless, to further extend the power of this important model organism requires a robust and simple-to-use genome editing platform that will enable targeted gene knockouts and introduction of specific mutations identified in human diseases into the zebrafish genome. We describe here protocols for creating insertion or deletion (indel) mutations or precise sequence modifications in zebrafish genes using customizable CRISPR-Cas9 RNA-guided nucleases (RGNs). These methods can be easily implemented in any lab and may also potentially be extended for use in other organisms.

Cas9-based genome editing in zebrafish.

Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unprecedented efficacy and facility in a wide variety of biological systems. In zebrafish, specifically, studies have shown that Cas9 can be directed to user-defined genomic target sites via synthetic guide RNAs, enabling random or homology-directed sequence alterations, long-range chromosomal deletions, simultaneous disruption of multiple genes, and targeted integration of several kilobases of DNA. Altogether, these methods are opening new doors for the engineering of knock-outs, conditional alleles, tagged proteins, reporter lines, and disease models. In addition, the ease and high efficiency of generating Cas9-mediated gene knock-outs provides great promise for high-throughput functional genomics studies in zebrafish. In this chapter, we briefly review the origin of CRISPR/Cas technology and discuss current Cas9-based genome-editing applications in zebrafish with particular emphasis on their designs and implementations.