Structural insights into RNA interference

Virtually all animals and plants utilize small RNA molecules to control protein expression during different developmental stages and in response to viral infection. Structural and mechanistic studies have begun to illuminate three fundamental aspects of these pathways: small RNA biogenesis, formation of RNA-induced silencing complexes (RISCs), and targeting of complementary mRNAs. Here we review exciting recent progress in understanding how regulatory RNAs are produced and how they trigger specific destruction of mRNAs during RNA interference (RNAi)

Mechanism of Foreign DNA Selection in a Bacterial Adaptive Immune System

In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary "nonself" DNA sequences for destruction, while avoiding binding to "self" sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for nonself target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved three base pair motif that is required for nonself target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade

CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution

High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high-throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.This is a pre-print of the article Murugan, Karthik, Arun S. Seetharam, Andrew J. Severin, and Dipali G. Sashital. "High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants." bioRxiv (2020). DOI: 10.1101/2020.05.12.091991. Attribution 4.0 International (CC BY 4.0). The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Posted with permission

Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

Cas12a (formerly Cpf1) is an RNA-guided endonuclease in the CRISPR-Cas immune system that can be easily programmed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for precise genome editing applications. This specificity is contradictory to the natural role of Cas12a as an immune effector against rapidly evolving phages. However, the native cleavage specificity and activity remains to be fully understood. We employed high-throughput in vitro cleavage assays to determine and compare the native specificities of three Cas12a orthologs. Surprisingly, we observed pervasive nicking of randomized target libraries, with strong nicking activity observed against targets with up to four mismatches. Nicking and cleavage activities are dependent on mismatch type and position, and vary depending on the Cas12a ortholog and crRNA sequence. Our high-throughput and biochemical analysis further reveal that Cas12a has robust activated non-specific nicking and weak non-specific dsDNA degradation activity in trans. Together, our findings reveal Cas12a cleavage activities that could be beneficial in the context of bacterial CRISPR-Cas immunity but may be detrimental for genome editing technology.This is a pre-print of the article Murugan, Karthik, Arun S. Seetharam, Andrew J. Severin, and Dipali G. Sashital. "Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a." bioRxiv (2019): 657791. DOI: 10.1101/657791. Attribution 4.0 International (CC BY 4.0). The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Posted with permission

Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade

CRISPR-Cas systems defend bacteria and archaea against infection by bacteriophage and other threats. The central component of these systems are surveillance complexes that use guide RNAs to bind specific regions of foreign nucleic acids, marking them for destruction. Surveillance complexes must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear. Here, we used single-molecule FRET to visualize how the type I-E surveillance complex Cascade searches DNA in real time. Cascade rapidly and randomly samples DNA through nonspecific electrostatic contacts, pausing at short PAM recognition sites that may be adjacent to the target. We identify Cascade motifs that are essential for either nonspecific sampling or positioning and readout of the PAM. Our findings provide a comprehensive structural and kinetic model for the Cascade target-search mechanism, revealing how CRISPR surveillance complexes can rapidly search large amounts of genetic material en route to target recognition