Investigation of Conformational Dynamics Involved in Genome Editing Events by CRISPR-Cpf1

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins are part of the adaptive immune system of bacteria and archaea. CRISPRs are stretches of DNA with two distinct characteristics: the presence of nucleotide repeats and spacers, which serve as a memory bank, enabling bacteria to recognize the invading viruses. The Cas proteins are enzymes that cut foreign DNA guided by the information encoded in CRISPRs. In 2012, it has been demonstrated that bacterial CRISPR-Cas9 can be transformed into a simple, programmable genome-editing tool. Cas9 requires two RNA molecules: a CRISPR RNA (or “crRNA”) and another tracrRNA (or “trans-activating crRNA”) to make its cut on a target DNA with a 20-nucleotide stretch complementary to the crRNA. Recently, Cpf1 proteins have been discovered to show comparable genome-editing capability to Cas9. Cpf1 functions through a single crRNA without an additional tracrRNA. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system, which is a smaller and simpler than Cas9, and overcomes some of the CRISPR/Cas9 system limitations. We are interested in rationally modifying the CRISPR-Cpf1 system to improve its genome editing efficiency. The target efficiency and specificity of the CRISPR technology are influenced by multiple factors. Our specific objective is to first perform large-scale molecular dynamics (MD) simulations to elucidate various conformational states involved in the Cpf1 catalysis (especially those upon the binding of crRNA and DNA) and the interconversion rates between different states. We further aim to use these MD simulations to uncover the molecular mechanisms underlying the higher genome editing efficiency of certain crRNAs. We will systematically explore how the incorporation of chemically and structurally modified nucleotides in crRNAs may affect the genome editing efficiency and off-target effects and to establish comprehensive structure-activity relationships of crRNA analogs.