Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing

CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction

A Systematic Analysis of Argonaute Proteins in Caenorhabditis elegans

RNA interference (RNAi) pathways, consisting of Argonaute proteins and small RNAs that provide sequence specificity, play key roles in gene regulation across all domains of life. Studies of Argonautes in many species have revealed that they impact gene expression at nearly every stage in the life cycle of a transcript—from transcription to translation. Due to their central role in RNAi and profound impact on development and differentiation in numerous organisms, uncovering new and conserved molecular mechanisms of Argonautes advances our fundamental knowledge of cellular function and has the potential to provide more precise means to manipulate gene expression, relevant for biotechnology and therapeutics.The C. elegans genome encodes an expanded family of 27 ago genes, 19 of which produce functional proteins. In this thesis I have undertaken a systematic study of every functional C. elegans Argonaute to develop a comprehensive portrait of the molecular mechanisms of these mostly uncharacterized proteins throughout development. Using CRISPR/Cas9 genome-editing, I epitope-tagged each Argonaute with GFP::3xFLAG. I used confocal microscopy to characterize the expression patterns of each Argonaute throughout development and defined the tissue specific and subcellular localizations of each Argonaute. High-throughput sequencing of 1) small RNAs associated with each Argonaute, and 2) total small RNA pools from argonaute mutants versus wild type uncovered the stratification of subsets of Argonautes into distinct gene regulatory modules. Directed by the localization and small RNA sequencing data, I employed phenotypic analyses of argonaute mutants under normal and stressful conditions. These revealed previously unappreciated phenotypes, including a transgenerational loss of fertility known as the Mortal Germline (mrt) defect, and resistance to the bacterial pathogen Pseudomonas aeruginosa. Overall, my systematic and pioneering studies provide an unprecedented view of the small RNA regulatory landscape throughout the development of a complex animal.Ph.D

A comprehensive survey of C. elegans argonaute proteins reveals organism-wide gene regulatory networks and functions

Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode Caenorhabditis elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work, we systematically analyzed every C. elegans AGO using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss