Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs.

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-an early step in NHEJ-yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion

Studies in Theoretical Biology: A Collectionof Undergraduate Research

120 pages, 7 articles*Studies in Theoretical Biology: A Collectionof Undergraduate Research* (Castillo-Chavez, Carlos) 120 pages*A Model Describing the Response of the Immune System to Mycobacterium tuberculosis* (Herrera, Christian; Lima, Sharon; Munoz, Roberto; Ramos, Gloria; Rodriguez, Ariel; Salzberg, Claudia) 19 pages*Mathematical Models to Study the Outbreaks of Ebola* (Astacio, Jaime; Briere, DelMar; Guillen, Milton; Martinez, Josue; Rodriguez, Francisco; Valenzuela-Campos, Noe) 18 pages*Stochastic Simulations of a Spatial SIR Model* (Camacho, Judit; Carreon, Fernando; Castillo-Guajardo, Derik; Jimenez-Perez, Hugo; Montoya-Gallardo, Leticia; Saenz, Ricardo A.) 14 pages*HIV-1 Replication Rate* (Arias, Michelle J.; Inguez, Delmy; Camacho, Erika T.; Castillo, Rafael B.; Melon, Eliel; Parra, Luz E.) 20 pages*The Effects of Vaccination in a Core Group* (Bobadilla, Marina; Lozano, Sharon Ann; Maia, Jessica Mendes; Villarreal, Julio Casar; Wilson, Novaline Dawn; Winston, Roberta) 19 pages*A Mathematical Model of the Dynamics of Rickettsia rickettsii in Tick-Host Interaction* (Alderete, Mary E.; Castillo-Garsow, Carlos W.; Salivia, Guarionex Jordan; Lara-Moreno, Carlos F.; Ramirez, Gina F.; Yichoy, Monica F.) 18 page