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Abstract CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9) has been revolutionizing genome engineering, and in-depth understanding of mechanisms governing its DNA discrimination is critical for continuing technology advances. An arginine-rich bridge helix (BH) connecting the nuclease lobe and the recognition lobe, which is conserved across the Cas9 family, exists in a helix–loop–helix conformation in the apo wild-type protein but converts to a long contiguous helix in the Cas9/RNA binary complex. In this work, distances measured with spin labels were utilized to investigate BH’s conformational transitions in the solution state upon single-guide RNA (sgRNA) binding, which is a critical early event preceding DNA binding and cleavage. Analyses show that sgRNA binding drives BH conformational changes in the wild-type SpyCas9 (SpyCas9WT) as well as in two BH-loop variants, SpyCas92Pro and SpyCas92Ala. Each Cas9–sgRNA binary complex, however, exhibits distinct BH features that reveal mutation-specific effects on helical integrity versus side-chain interactions. In addition, the BH conformational variations can be correlated to the observed changes in the mismatch cleavage profiles of the Cas9 variants. The work represents the first use of distances measured by site-directed spin labeling to investigate Cas9 protein conformational changes in the solution state and advances our understanding on the structure–dynamic–function relationship governing DNA target discrimination by Cas9. 

ABSTRACT CRISPR-Cas12a is widely used for genome editing and biomarker detection since it can create targeted double-stranded DNA breaks and promote non-specific DNA cleavage after identifying specific DNA. To mitigate the off-target DNA cleavage of Cas12a, we previously developed aFrancisella novicidaCas12a variant (FnoCas12a^KD2P) by introducing double proline substitutions (K969P/D970P) in a conserved helix called the bridge helix (BH). In this work, we used cryogenic electron microscopy (cryoEM) to understand the molecular mechanisms of BH-mediated activation of Cas12a. We captured five structures of FnoCas12a^KD2Pat different states of conformational activation. Comparison with wild-type (FnoCas12a^WT) structures unravels a mechanism where BH acts as a trigger that allosterically activates REC lobe movements by tracking the number of base pairs in the growing RNA-DNA hybrid to undergo a loop-to-helical transition and bending to latch onto the hybrid. The transition of the BH is coupled to the previously reported loop-to-helix transition of the “lid”, essential for opening RuvC endonuclease, through direct interactions of residues of the BH and the lid. We also observe structural details of cooperativity of BH and “helix-1” of RuvC for activation, a previously proposed interaction. Overall, our study enables development of high-fidelity Cas12a and Cas9 variants by BH-modifications. 

Bacterial and archaeal CRISPR-Cas systems offer adaptive immune protection against foreign mobile genetic elements (MGEs). This function is regulated by sequence specific binding of CRISPR RNA (crRNA) to target DNA/RNA, with an additional requirement of a flanking DNA motif called the protospacer adjacent motif (PAM) in certain CRISPR systems. In this review, we discuss how the same fundamental mechanism of RNA-DNA and/or RNA-RNA complementarity is utilized by bacteria to regulate two distinct functions: to ward off intruding genetic materials and to modulate diverse physiological functions. The best documented examples of alternate functions are bacterial virulence, biofilm formation, adherence, programmed cell death, and quorum sensing. While extensive complementarity between the crRNA and the targeted DNA and/or RNA seems to constitute an efficient phage protection system, partial complementarity seems to be the key for several of the characterized alternate functions. Cas proteins are also involved in sequence-specific and non-specific RNA cleavage and control of transcriptional regulator expression, the mechanisms of which are still elusive. Over the past decade, the mechanisms of RNA-guided targeting and auxiliary functions of several Cas proteins have been transformed into powerful gene editing and biotechnological tools. We provide a synopsis of CRISPR technologies in this review. Even with the abundant mechanistic insights and biotechnology tools that are currently available, the discovery of new and diverse CRISPR types holds promise for future technological innovations, which will pave the way for precision genome medicine. 

The DNA inside human cells provides instructions for all of the processes that happen inside the body. Errors in the DNA may lead to cancer, sickle cell disease, cystic fibrosis, Huntington’s disease, or other genetic disorders. Medical researchers are exploring whether it is possible to replace or repair the faulty DNA (an approach known as gene therapy) to reduce the symptoms, or even cure individuals, of these conditions. Over the last ten years, a new technology known as CRISPR-Cas9 gene editing has proved to be a reliable and efficient way to make small and precise changes to DNA in living cells. First, an enzyme called Cas9 searches for a segment of target DNA segment that matches a template molecule the enzyme carries. Cas9 then cuts the target DNA, which is repaired to match a new customized DNA sequence: this changes the genetic information of the cell. The Cas9 protein is made of a succession of building blocks called amino acids that create long chains which then fold to form the final three-dimensional shape of the enzyme. A region of Cas9 known as the HNH domain is responsible for cutting the target DNA. However, it remains unclear exactly which amino acids within this domain work together to sever the DNA. Here, Zuo et al. combined computational and experimental approaches to reveal the three-dimensional structure of the Cas9 enzyme when the HNH domain is poised to cut the target DNA. The findings were used to generate a computational model of Cas9 and this model predicted that the HNH domain relies on a group of three amino acids known collectively as D839-H840-N863 to cleave DNA strands. This knowledge is useful to understand exactly how Cas9 modifies genetic information. Ultimately, this may help to improve CRISPR-Cas9 technology so it could be safely used in geneediting therapies.