Using a chemically-controlled CRISPR/Cas9 system to understand and develop new genome engineering technologies

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) systems have revolutionized our ability to investigate the genotype-phenotype relationships of specific genetic elements. The class 2 type II CRISPR system, involving a Cas9 endonuclease, has been widely adopted to aid in making specific genomic DNA changes. Precise DNA targeting by the CRISPR/Cas9 system is achieved using an RNA molecule that encodes a 20 nucleotide (nt) sequence complementary to the target site. Cas9 can be targeted to different loci in the genome by simply changing the 20 nt RNA-encoded sequence. Cas9 can then create DNA double-strand breaks (DSBs) at the target site and induce DNA repair to incorporate a specific DNA edit or uncontrolled insertions and deletions to knock out a gene. The CRISPR/Cas9 system has also been adapted to create a precise DNA targeting module that can recruit different DNA effector systems, such as transcriptional activators, DNA deaminases, and histone modifiers. While CRISPR/Cas9 has enabled new insights into genotype-phenotype relationships, challenges remain with the formation of unwanted edits, such as off-target edits or bystander edits with base editor systems. Furthermore, there is a lack of a generalizable method to create temporally-controlled Cas9-based effector systems to allow investigation of temporally-regulated genetic elements. Here, I use a chemically-inducible Cas9 (ciCas9) to explore the in vivo mechanisms of Cas9 off-target editing and to develop a generalizable system to confer temporal control over a variety of Cas9-based effector systems. Using engineered chemically-controlled base editors, I dissected the kinetics of bystander editing and how base editing at one nucleotide influences subsequent base edits within the same target site. I envision the results presented here could be used to inform future efforts to study temporally-regulated genetic elements and to engineer more efficient and accurate Cas9-based genome engineering systems.