Protein Engineering of Cas9 for Enhanced Function

Abstract

CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complimentary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted directly into the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome modifying tools.

mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence

“…Changes to the stem-loop architecture of the tracrRNA portion of a sgRNA greatly strengthen its affinity for Cas9 ( Chen et al 2013 ), and shortening of the crRNA portion of a sgRNA to just 20 nucleotides reduces off-target action while preserving efficiency ( Pattanayak et al 2013 ). The range of DNA/chromosome-based applications has been further extended by engineering of S. pyogenes Cas9 [or use of Cas9 orthologs from other bacterial species ( Jinek et al 2014 )] to relax its requirement for initiating DNA sequence recognition at a so-called PAM (“protospacer adjacent motif”) site (5′-NGG-3′) ( Kleinstiver et al 2015 ), to inactivate one or both of its two (McrA/HNH-like and RuvC/RNAaseH-like) catalytic sites to create a “nickase” ( Fu et al 2014 ) or a catalytically “dead” (dCas9) version ( Gilbert et al 2013 ), or to insert new functionalities ( Oakes et al 2014 ). Cas9 and associated sgRNAs have been used in diverse organisms for genome editing, both gene knock-outs ( Gaj et al 2013 ) and gene fusions ( Wei et al 2013 ), as well as to force biased inheritance of a desired allele within entire populations (“gene drives”) ( DiCarlo et al 2015 ; Dong et al 2015 ; Gantz et al 2015 ).…”

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch

“…A PDZ domain with flanking amino acid linkers was cloned into the naïve library ( Supplementary Fig 1 , Supplementary Fig. 2 ) and passaged through two rounds of a CRISPRi screen 18 , 19 . Briefly, cells expressing Red Fluorescent Protein (RFP) and Green Fluorescent Protein (GFP) were assayed using fluorescence activated cell-sorting (FACS) to identify Cas9 variants capable of repressing RFP in a single guide RNA (sgRNA)-dependent fashion ( Supplementary Fig 4 ) 18 , 19 .…”

RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

“…Asymmetric distributions have been found in several previous targeting studies, for example a study using the ISY100 transposon (which, like SB, is a member of the Tc1/mariner transposon superfamily) in combination with the ZF domain Zif268 in E. coli (76) or in vitro experiments with dCas9/Hsmar1 fusions (55). Enrichment mainly occurring downstream of the sgRNA target site was somewhat surprising, as domains fused to the Cterminus of Cas9 are expected to be localized closer to the 5'-end of the target strand (77), or upstream of the sgRNA binding site. The fact that SB100X is connected with dCas9 by a relatively long, flexible linker could explain why enrichment can occur on the other side of the sgRNA binding site, but it does not explain why enrichment on the 'far side' seems to be more efficient.…”