Genome oligopaint via local denaturation fluorescence in situ hybridization.

Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue.

Real-time observation of Cas9 postcatalytic domain motions.

CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3' flap generated by NTS cleavage can be rapidly digested by a 3' to 5' single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3' flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9's conformation and suggest possible biotechnological applications.

Manipulating the revision of reward value during the intertrial interval increases sign tracking and dopamine release.

Recent computational models of sign tracking (ST) and goal tracking (GT) have accounted for observations that dopamine (DA) is not necessary for all forms of learning and have provided a set of predictions to further their validity. Among these, a central prediction is that manipulating the intertrial interval (ITI) during autoshaping should change the relative ST-GT proportion as well as DA phasic responses. Here, we tested these predictions and found that lengthening the ITI increased ST, i.e., behavioral engagement with conditioned stimuli (CS) and cue-induced phasic DA release. Importantly, DA release was also present at the time of reward delivery, even after learning, and DA release was correlated with time spent in the food cup during the ITI. During conditioning with shorter ITIs, GT was prominent (i.e., engagement with food cup), and DA release responded to the CS while being absent at the time of reward delivery after learning. Hence, shorter ITIs restored the classical DA reward prediction error (RPE) pattern. These results validate the computational hypotheses, opening new perspectives on the understanding of individual differences in Pavlovian conditioning and DA signaling.

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding.

Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5' end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5' end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a).

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation.

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.

Salactin, a dynamically unstable actin homolog in Haloarchaea.

IMPORTANCE: Protein filaments play important roles in many biological processes. We discovered an actin homolog in halophilic archaea, which we call Salactin. Just like the filaments that segregate DNA in eukaryotes, Salactin grows out of the cell poles towards the middle, and then quickly depolymerizes, a behavior known as dynamic instability. Furthermore, we see that Salactin affects the distribution of DNA in daughter cells when cells are grown in low-phosphate media, suggesting Salactin filaments might be involved in segregating DNA when the cell has only a few copies of the chromosome.

Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis.

Cas9 (from Streptococcus pyogenes) in complex with a guide RNA targets complementary DNA for cleavage. Here, we developed a single-molecule FRET analysis to study the mechanisms of specificity enhancement of two engineered Cas9s (eCas9 and Cas9-HF1). A DNA-unwinding assay showed that mismatches affect cleavage reactions through rebalancing the unwinding-rewinding equilibrium. Increasing PAM-distal mismatches facilitates rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily with mismatches. The intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. Engineered Cas9s require approximately one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity by inhibiting stable DNA binding to partially matching sequences, making DNA unwinding more sensitive to mismatches and slowing down the intrinsic cleavage reaction.