Screening highly-specific hairpin guide RNAs for CRISPR-Cas gene editing

This thesis outlines the development of a screening method to improve the specificity of the CRISPR effector, Cas9, in gene editing applications. We accomplish this by identifying modifications to the RNA-cofactor of Cas9, its guide RNA (gRNA), that determines the DNA sequence recognized by the effector, where changes to the gRNA secondary structure are hypothesized to limit the potential for mutations at unintended sites. The identification of highly specific gRNA sequences can potentially lead to highly specific gene editing techniques that can be used to treat genetic diseases such as Tay-Sachs disease, cystic fibrosis, and sickle cell anemia without off-target mutations. Randomized libraries of gRNAs with extra nucleotides that can form secondary structures such as “hairpins” with the targeting segment of the gRNA (hairpin-gRNAs or hp-gRNAs) are screened with SpyCas9 in the presence of DNA with the intended target, and DNA containing “off-target” sequences that are similar but not an exact match for the target. The results from this screen can provide insights to the biophysical requirements that dictate target recognition and the potential for design rules for high-fidelity hp-gRNAs for any genetic target for use in effective gene therapeutics

Selection of Extended CRISPR RNAs with Enhanced Targeting and Specificity

ABSTRACTFor a CRISPR guide RNA (gRNA) with a specific target but activity at known “off-target” sequences, we present a method to screen hundreds of thousands of gRNA variants with short, randomized 5’ nucleotide extensions near its DNA-targeting segment—a modification that can increase Cas9 gene editing specificity by orders of magnitude with certain 5’- extension sequences,viasome as-yet-unknown mechanism that makesde novodesign of the extension sequence difficult to perform manually—to robustly identify extended gRNAs (x-gRNAs) that have been counter-selected against activity at those off-target sites and that exhibit significantly enhanced Cas9 specificity for their intended targets.</jats:p

Elution and uncoating of Coxsackievirus B3 by isolated HeLa cell plasma membranes

Plasma membranes isolated from HeLa cells on discontinuous sucrose gradients were assayed for their capacity to elute and uncoat coxsackievirus B3 at 37 C. Because the viral receptors are limited to the surface of HeLa cells, the addition of radioactively labeled virus to the cells prior to cell homogenization provided a useful marker for locating the plasma membranes during the fractionation procedure. Four bands were formed on the discontinuous sucrose gradients with approximately 70% or more of the membrane-associated viral label being recovered in the most dense bands, designated as bands 3 and 4. Bands 3 and 4 also possessed the plasma membrane marker enzymes, Na+, K+ adenosine triphosphatase and 5'-nucleotidase and revealed typical structures characteristic of plasma membranes as revealed by electron microscopy. Pelleted and washed membranes from band 3 both eluted and uncoated B3 32P-labeled virus, whereas membranes from band 4 eluted virus but failed to uncoat it. The membranes from band 4 were shown to inhibit the viral uncoating activity when mixed with membranes of band 3. Characteristically, unfractionated homogenates of cell membranes eluted but did not uncoat virus. The finding of a naturally occurring inhibitor of virus uncoating provides for the first time a way to distinguish between the membrane activities of virus elution and virus uncoating. The inhibitor remains to be characterized.</jats:p