Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells

Abstract

Engineering and study of protein function by directed evolution has been limited by the requirement to introduce DNA libraries of defined size or to use global mutagenesis. Here, we develop a strategy to repurpose the somatic hypermutation machinery used in antibody affinity maturation to efficiently perform protein engineering in situ. Using catalytically inactive Cas9 (dCas9) to recruit variants of the deaminase AID (CRISPR-X), we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations, in contrast to insertions and deletions created by active Cas9, and can be used to mutagenize multiple genomic locations simultaneously. With this technology, we mutagenize GFP and select for spectrum-shifted variants, including EGFP. In addition, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer resistance to treatment. Finally, we utilize a hyperactive AID variant with dramatically increased activity to mutagenize endogenous loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful new approach to create highly complex libraries of genetic variants in native context, which can be broadly applied to investigate and improve protein function.

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

“…This is similar to the process of somatic hypermutation during antibody affinity maturation, normally performed by AID. Two technologies, Targeted AID-mediated Mutagenesis (TAM) (Ma et al, 2016) and CRISPR-X (Hess et al, 2016), use this strategy to generate localized sequence diversity through base editing.…”

“…In the CRISPR-X system (Hess et al, 2016) dCas9 is used to recruit a hyperactive variant of the AID enzyme. Three mutations (K10E, T82I, and E156G) were previously identified in a bacterial screen that increased the deamination activity of AID (Wang et al, 2009); this variant was combined with a deletion of the NES to generate AID*Δ.…”

High-Throughput Approaches to Pinpoint Function within the Noncoding Genome

“…Base editing can be performed by the fusion of a cytidine deaminase enzyme, such as rat-derived APOBEC1, to catalytically inactive Cas9 (dCas9) (Komor et al, 2016) (Figure 2B). Other cytidine deaminase enzymes, such as activation-induced cytidine deaminase (AID), have also been used (termed “CRISPR-X”) (Hess et al, 2016; Nishida et al, 2016). These fusions allow for modification of C→T or G→A in the absence of a DSB, with mutation predominantly occurring in a 3- to 5-bp window, which was recently narrowed to 1 to 2 bp for enhanced targeting specificity (Kim et al, 2017c).…”

“…Disruption of CTCF binding sites can act as an oncogenic driver via enhancer hijacking and activation of neighboring oncogenes (Hnisz et al, 2016). Several tools exist for CTCF manipulation, such as nucleases for mutagenesis sterically hindering CTCF binding (CRISPRi), methylation of CpG dinucleotides (dCas9-DNMT3A, dCas9-MQ1), and base editing approaches such as cytosine deaminase effectors (Hess et al, 2016; Lei et al, 2017; Liu et al, 2016; Ma et al, 2016). Combining these tools with a genome-wide CTCF library would allow rapid interrogation of the CTCF sites and TADs that are most relevant for particular diseases and phenotypes.…”

An optimized toolkit for precision base editing

“…Base editors are hybrid proteins that tether DNA modifying enzymes to nuclease defective Cas9 variants. This enables the direct conversion of cytosine (C) to other bases (T, A, or G) [1][2][3][4] , or adenine (A) to inosine/guanine (I/G) nucleic acids 5 6 , enabling the creation or repair of disease-associated single nucleotide variants (SNVs), or molecular recording 7 . The BE3 base editor carries a rat APOBEC cytidine deaminase at the N-terminus of Cas9n (Cas9 D10A ) and a uracil glycosylase inhibitor (UGI) domain at the C-terminus.…”