CRISPR-CAS9-MEDIATED GENOME EDITING

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are required for bacterial and archeal innate immunity against foreign DNA (Marraffini, Nature, 2015). During the acquisition phase, foreign DNA fragments are incorporated into CRISPR loci located in the bacterial and archeal genome. Transcribed RNA from CRISPR loci is subsequently processed into CRISPR RNAs, which are then incorporated into Cas proteins. The most studied Cas protein is Cas9, a DNA endonuclease that forms a complex with CRISPR RNAs to cleave foreing DNA at specific sequences recognized by CRISPR RNAs (Figure 8) (Jinek et al, Science, 2012). CRISPR-Cas9 has been engineered to introduce sequence specific DNA double-strand breaks (DSBs) in all cellular and animal models currently studied, therefore allowing site-specific genome editing by homology-directed repair (HDR) or non-homologous end joining (NHEJ) (Wright et al, Cell, 2016Hsu et al, Cell, 2014).

Figure 8. CRISPR-Cas9-mediated DNA double-strand break formation and repair

INACTIVATION OF EUKARYOTIC GENES BY INDUCTION OF STOP CODONS (iSTOP)

As an alternative to HDR- and NHEJ-dependent genome editing, CRISPR-dependent editing strategies that entail direct modification of DNA bases have recently been developed (Hess et al, Nature Methods, 2016; Komor et al, Nature, 2016;  Ma et al, Nature Methods, 2016; Nishida et al, Science, 2016; Yang et al, Nature Comm, 2016). Distinct from standard CRISPR-Cas9-dependent genome editing, CRISPR-mediated base editing avoids the formation of DSBs, thus resulting in reduced genomic rearrangements and cell death (Kuscu et al, Nature Methods, 2017). CRISPR-dependent base editors consist of a catalytically inactive form of Cas9 or a Cas9 nickase mutant fused to cytidine deaminases, such as APOBEC1 or AID. In particular, the CRISPR-dependent base editor BE3 is a fusion of rat APOBEC1 (rAPOBEC1), a uracil glycosylase inhibitor (UGI) and the Cas9-D10A nickase mutant (Komor et al, Nature, 2016; Figure 9). Our studies have shown that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA and TGG) into STOP codons without DSB formation (Billon, Bryant et al, Mol Cell, 2017; Figure 9). To facilitate gene inactivation by induction of STOP codons (iSTOP), we have generated a database of over 3.4 million sgRNAs for iSTOP (sgSTOPs) targeting 97-99% of genes in 8 eukaryotic species (http://www.ciccialab-database.com/istop). This database includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay and restriction enzymes that allow the rapid detection of iSTOP-mediated editing in cell populations and clones. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. This work provides a comprehensive resource for DSB-free gene disruption by iSTOP.

Figure 9. Induction of STOP codons by CRISPR-mediated base editing

CURRENT STUDIES

Definition of cellular pathways that regulate CRISPR-mediated gene editing

We are currently employing genetic and biochemical tools to define the cellular machineries that repair DSBs formed using CRISPR-Cas9 technology and identify factors that determine whether Cas9-induced DSBs are repaired by HDR or NHEJ. Furthermore, we are defining cellular pathways that modulate CRISPR-dependent base editing. These studies will provide novel insights into CRISPR-mediated gene editing and lead to the development of improved CRISPR-based technologies.