Single molecule analysis of CRISPR enzymes

CRISPR;Single Molecule;Biophysics;Gene Editing;Genome Engineering;Cas9;smFRET;Fluorescence;FRET;Biochemistry;Molecular Biology.;Biophysics

Singh, DigvijaySeydoux, Geraldine ;

Johns Hopkins University

关键词: CRISPR;    Single Molecule;    Biophysics;    Gene Editing;    Genome Engineering;    Cas9;    smFRET;    Fluorescence;    FRET;    Biochemistry;    Molecular Biology.;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/59155/SINGH-DISSERTATION-2018.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

CRISPR system provides adaptive immunity against foreign DNA in microbes. CRISPR nucleases in complex with a guide-RNA targets complementary DNA (protospacer) if they are next to protospacer adjacent motif (PAM), by the virtue of RNA-DNA base pairing, resulting in unwinding of DNA strands in protospacer. CRISPR has been re-purposed for wide-ranging biological applications. But lack of mechanistic understanding and specificity of both binding and cleavage continue to be challenges for CRISPR based applications. I employed single molecule fluorescence and biochemical assays to investigate the molecular mechanism of widely used CRISPR-enzymes (Cas9, its engineered derivatives (EngCas9) and Cpf1 orthologs). Following observations were made:• CRISPR-enzymes bind DNA in 2 modes. In mode I, enzymes samples DNA non-specifically for transient PAM-detection. Upon successful PAM-recognition, binding shifts to mode II involving RNA-DNA duplex/DNA unwinding. Mode-II is longer lived, whose lifetime depends on number and location of on/off-target base-pairs (bp) (PAM-proximal off-targets bp; deleterious PAM-distal ones; tolerable).• Cas9s require only 9-10 bp for stable binding and ~16 bp for cleavage. Cpf1s are significantly more specific, requiring ~17 bp for both. • Release of cleaved-DNA products for genome-editing machinery is likely crucial. Cas9s do not release any, where Cpf1s releases one end.• Cas9s cleavage happens from maximally unwound DNA state. PAM-distal off-target bp reduce extent and lifetime of unwinding thus delaying cleavage. EngCas9s mutations destabilize unwound state and have a much lower intrinsic cleavage rate, which are the basis of their improved specificity.• Significant effect of pH and reducing conditions on Cpf1 activity can explain higher performance variance (compared to Cas9) in different organisms. These can also be used as switches for Cpf1 activation/inactivation.The work presented in this dissertation has already resulted in 3 first-author manuscripts and a review. The work has been further extended into other fluorescent geometries and onto newer CRISPR enzymes. I am also applying the biophysical information from my PhD work to re-engineer CRISPR-Cas9 for precise control of its DNA binding and cleavage activity. These efforts have resulted in useful data and will likely lead to more manuscripts. Continued improvement in our understanding of CRISPR mechanism will assist in rational-design of new enzymes/strategies with higher specificity and efficiency.