BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF BACTERIAL RNA-GUIDED DNA TARGETING SYSTEMS

CRISPR;Genome editing;Cas9;Biochemistry

Chen, HongfanPrigge, Sean T. ;

Johns Hopkins University

关键词: CRISPR;    Genome editing;    Cas9;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/40726/CHEN-DISSERTATION-2016.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated proteins) immune systems found in many prokaryotes rely on small guide CRISPR RNAs (crRNAs) to destroy invading viruses and plasmids. This RNA-guided adaptive immune response is mediated by numerous diverse Cas proteins, several of which form complexes with crRNAs and function to silence foreign nucleic acids. Current understanding of the molecular basis of these proteins is limited. Here, we present biochemical and structural characterization of two sets of such proteins: Cascade from Escherichia coli and Cas9 from Streptococcus thermophilus. E. coli Cascade, a large multimeric ribonucleoprotein complex, uses crRNA to base pair with complementary DNA (protospacer) at sites adjacent to a signature sequence termed the protospacer adjacent motif (PAM). The bound structure, known as an R-loop, propagates from PAM to the other end of the protospacer. A crystal structure of Cascade bound to a ssDNA target, previously determined by our laboratory, reveals a potential pocket for binding of the displaced strand in the R-loop. Here we provide experimental evidence that this pocket serves as a docking site for the displaced strand, and this binding facilitates DNA strand separation during R-loop formation. Structure-guided mutagenesis of the basic residues in the pocket confirms their importance for double strand DNA binding. Single-molecule experiments reveal that these mutations kinetically hinder R-loop formation. We further show that Cascade exerts a strong conformational ;;lock” upon completion of an R-loop, and this locked conformation is sufficient for recruiting the trans-acting Cas3 helicase/nuclease for target destruction. Cas9 from S. thermophilus LMG18311 is 1122 amino acid protein harboring a HNH nuclease domain and a RuvC-like nuclease domain. We demonstrate that LMG18311 Cas9 utilizes a crRNA in conjunction with a trans-acting crRNA (tracrRNA) to cleave double strand DNA in vivo and in vitro. The cleavage is dependent on the presence of PAM as well as the position of the PAM. We further show that the HNH and RuvC-like nuclease domains of Cas9 select the location of their cleavage sites via different mechanisms. The HNH domain catalyzes cleavage of the target strand at a fixed position, whereas the RuvC-like domain catalyzes cleavage of the non-target strand using a ruler mechanism.