Catalytically Enhanced Cas9 through Directed Protein Evolution - PubMed (original) (raw)

Catalytically Enhanced Cas9 through Directed Protein Evolution

Travis H Hand et al. CRISPR J. 2021 Apr.

Abstract

Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIG. 1.

FIG. 1.

Selection of catalytically enhanced Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9). (A) Top: Domain organization of AceCas9 with amplified view of linker II (L-II) with primers used for error-prone polymerase chain reaction primers. Bottom: Directed protein evolution selection strategy. pACYC-AceCas9-g123 and pACYC-AceCas9-g119 co-express AceCas9 and the single-guide RNA with 24mer and 20mer guide, respectively. (B) Sequence logo of consensus sequence from next-generation sequencing analysis of the final pool. The height of each letter is proportional to the observed frequency of each nucleotide in the alignment column. The locations with highest deviation are marked by their corresponding amino acid positions. (C) Pie charts illustrate enrichment of amino acids at the four highest frequency sites. Pool 0 reflects enrichment values in the starting library, whereas pool 5 reflects those from the last round of survival colonies. Single letters for amino acids are used. Color images are available online.

FIG. 2.

FIG. 2.

Verification of the catalytically enhanced AceCas9 by survival assay and kinetic analysis. (A) Plasmids encoding the selected AceCas9 mutants were transformed into ccdB-harboring cells that are plated on plates with (Ara+) or without arabinose (Ara–). Images of the plates are shown with the corresponding survival rate plotted below. (B) Single-turnover DNA cleavage experiments and the resulting rates of the AceCECas9 with either the 20mer spacer or the 24mer spacer single-guide RNA (sgRNA). Molar excess of AceCas9–sgRNA complex were incubated with plasmid DNA for various times at 50°C before being resolved and visualized on agarose gels. Kinetic experiments were performed in triplicate (Supplementary Fig. S4).

FIG. 3.

FIG. 3.

DNA binding competition assay results. The DNA oligo substrates used are schematically shown as wild-type (WT), T(-1)G/A(+1)C (dsDNA with mismatch to sgRNA at position +1), T(-1)T/A(+1)C (bulged DNA oligo at position +1 that mismatches with sgRNA), and non-target strand bulge (bulged DNA oligo at position at +1 that matches sgRNA). For each competition binding experiment, the cleavage gel image and fraction of cleavage versus competitor concentration at logarithmic scale are shown. Solid curves are fitted theoretical curves and fitted KI values are indicated. (A) Competition binding result for the wild-type or T(-1)G oligo with AceCas9 (WT) and the V709A variant. (B) Competition binding result for the bulged oligos with AceCas9 (WT) and the V709A variant. Experiments performed in triplicate (Supplementary Fig. S5). Color images are available online.

FIG. 4.

FIG. 4.

Gene insertion facilitated by the wild-type and the catalytically enhanced Streptococcus pyogenes Cas9 (SpyCas9) in HCT116 cells. (A) Cartoon illustration depicting the genome insertion process. The px330 vector co-expressing either the wild-type SpyCas9 or the SpyCECas9 with the sgRNA targeting the LMNB1 gene is co-transfected with the repair vector containing mEGFP flanked by 1 kb homology arms (HAs). Each transfection is performed with the Lonza SE cell line 4-D nucleofector solution kit by electroporating one million HCT116 cells in an electro-cuvette. Cells are plated and cultured for 48 h post transfection (positive cells are depicted as gray with a green ring around the nuclear lamina). The HCT116 cells that successfully achieved homology directed repair (HDR) by incorporation of the green fluorescent protein (GFP) are detected, sorted, collected via flow cytometry, and expanded for imaging. (B) Detailed scheme of the CRISPR-Cas9-mediated GFP tagging strategy. (1) The crRNA (purple) co-expressing with the wild-type SpyCas9 (px330, Addgene #42230) or the catalytically enhanced (CE) SpyCECas9 on a modified px330 vector targets a site upstream of exon 1 (red) of the LMNB1 gene. (2) A repair vector containing 1 kb HAs flanking the GFP-encoding sequence fused with a linker sequence (AICSDP-10:LMNB1-mEGFP, Addgene #87422). (3) Successfully edited target locus following SpyCas9 cleavage and HDR contains an in-frame insertion of the GFP after the start codon of LMNB1 exon 1. (C) The observed GFP signal of the wild-type and the catalytically enhanced SpyCas9. Flow cytometry plots displaying GFP fluorescence intensity (_x_-axis) versus RFP fluorescence intensity (_y_-axis; to control for auto-fluorescence) following 48 h post transfection for the wild-type (SpyCas9) and catalytically enhanced (SpyCECas9). GFP+ cells fall within the P2 gate. The negative control is obtained from cells transfected with the repair vector and SpyCas9 without a sgRNA. Experiments were conducted in triplicate (Supplementary Figure S7). (D) Bar graph displaying the averages of the quadruplicate measurement for both SpyCas9 and SpyCECas9, and error bars displaying standard deviation. Statistical significance was determined by an unpaired two-tailed _t_-test (*p ≤ 0.05, exact value shown below the asterisk). (E) Representative fluorescence microscopy images of GFP-tagged LMNB1 in HCT116 cells obtained on a DeltaVision (GE Life Sciences) microscope. The cell nuclei were stained with DAPI. Color images are available online.

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