PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation

Y Dhingra, Shravanti Krishna Suresh, P Juneja, DG Sashital

Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these “prespacer” substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4- PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation.

Beginner’s guide to CRISPR-Cas9-based gene editing

Shravanti K Suresh

In the last 20 years since its discovery, clustered regularly interspaced short palindromic repeats (CRISPR) has evolved from a mere bacterial immune system to a tool that can be programmed to achieve directed, efficient and sequence-specific modifications to the host genome. Its ease of use and minimum side effects to the cell lines are also a reflection of its ability to be extrapolated to medical environments, where it can be used for genetic modifications in the more complicated human genome. In research labs, CRISPR is now frequently used to achieve fast adjustments to host cell lines. It is used to knock in or knock out genes of interest and for generating models for genetic studies. In this guide I seek to help readers understand the basic requirements of a genome editing experiment using CRISPR-Cas components.

In situ modeling of multimodal floral cues attracting wild pollinators across environments

Karin Nordström, Josefin Dahlbom, V. S. Pragadheesh, Suhrid Ghosh, Amadeus Olsson, Olga Dyakova, Shravanti Krishna Suresh, and Shannon B. Olsson

The coeveolution of flowers and pollinators is well known, but how generalist pollinators identify suitable flowers across environments and flower species is not well understood. Hoverflies, which are found across the globe, are one of the most important alternative pollinators after bees and bumblebees. Here we measured, predicted, and finally recreated multimodal cues from individual flowers visited by hoverflies in three different environments (hemiboreal, alpine, and tropical). We found that although “flower signatures” were unique for each environment, some cues were ubiquitously attractive, despite not resembling cue combinations from real flowers. Our results provide unique insights into how a cosmopolitan pollinator identifies flower objects across environments, which has important implications for our understanding of pollination as a global ecological service.

Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants 

Karthik Murugan, Shravanti K Suresh, Arun S Seetharam, Andrew J Severin, Dipali G Sashital

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used 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. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically 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 could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.

Enzymatic anti-CRISPRs improve the bacteriophage arsenal

Shravanti K. Suresh, Karthik Murugan & Dipali G. Sashital

Bacteriophage-encoded anti-CRISPR (Acr) proteins were previously thought to inhibit CRISPR-mediated immunity by acting as physical barriers against the binding or cleavage of DNA. Two new studies report that recently discovered type V Acr proteins use enzymatic activities to shut down the Cas12a endonuclease, providing a multi-turnover ‘off switch’ for CRISPR-based immunity and technology.