Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila - PubMed (original) (raw)

. 2014 Nov 6;9(3):1151-62.

doi: 10.1016/j.celrep.2014.09.044. Epub 2014 Oct 23.

Zhihao Yang 1, Jiang Xu 2, Jin Sun 1, Decai Mao 3, Yanhui Hu 4, Su-Juan Yang 1, Huan-Huan Qiao 1, Xia Wang 1, Qun Hu 5, Patricia Deng 6, Lu-Ping Liu 7, Jun-Yuan Ji 8, Jin Billy Li 9, Jian-Quan Ni 10

Affiliations

Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila

Xingjie Ren et al. Cell Rep. 2014.

Abstract

The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. The criteria for off-target effects in the DGSC system

(A) Schematic showing the Cas9/sgRNA system. Cas9 is shown as the red background. The sgRNA is shown in green. The 3-nucleotide PAM sequence (NGG or NAG) in the DNA is shown in blue. The PAM-distal and – proximal ends are labeled by the green arrows. (B) Mutagenesis efficiency of sgRNAs with mismatches (red) to a target of the white locus. Results are from three independent experiments, and error bar shows standard error of the mean. (C) Mutagenesis efficiency of sgRNAs with mismatches (red) to targets of different loci. (D) Off-target mutagenesis efficiency of 10 sgRNAs (numbered 1 to 10), compared with the on-target efficiency. The potential off-targets have three nucleotide mismatches (red) to the sgRNAs. PAMs are indicated in bold text. (E-F) Mutagenesis efficiency of sgRNAs with 20 nt targeting sequences, those with 18 nt, and those with 18 nt sequences and one-base mismatches (red). (G-H) Mutagenesis efficiency of sgRNAs with targeting sequences ranging from 17 to 22 nt. The regular 20 nt sgRNAs are shown in blue. Each row in (A-B) and (E-H) represents an sgRNA sequence, and its mutagenesis rate. Each row in (C-D) represents a site of the fly genome, and the mutagenesis rate at that site. See also Figure S1.

Figure 2

Figure 2. Parameters affecting sgRNA mutagenesis efficiency

(A) Heritable mutation rate of sgRNA w1 (n = 3) and w2 (n = 3). (B) Fertile G0 rate of w1 (n = 3) and w2 (n = 3). Fertile G0 rate is calculated as the number of fertile G0 flies divided by total embryos injected. (C) Relative mutagenesis efficiency of w1 and w2. To calculate relative mutagenesis efficiency, heritable mutation rate is multiplied by fertile G0 rate and divided by the highest value. (A-C) The X-axis shows injection concentration of sgRNA vector at 0, 10, 25, 50, 75, 100, 150, 200, and 250 ng/μL. Results are from three independent experiments each for w1 and w2. Error bar shows SEM. (D) Plot figure showing the correlation between mutagenesis efficiency and sgRNA GC content, when different numbers of PAM-proximal nucleotides are considered. (E) Scatter plot demonstrating the correlation between sgRNA heritable mutation rate and 6 PAMPN GC content. Data points for the sgRNA targets in white (w, n = 27), vermilion (v, n = 4), ebony (e, n = 4), and yellow (y, n = 4) are shown. Pearson’s correlation coefficient (r) = 0.675. (F) A three-dimensional column graph that shows the comparison of correlation coefficient when different consecutive regions of the sgRNAs are considered. Each column represents the Pearson’s r between mutagenesis efficiency and GC content of a specific region of the sgRNAs. The 20-nt targeting sequence of the sgRNA is numbered with the nucleotide closest to PAM as 1 and the one farthest to PAM as 20. The x-axis shows the position of the nucleotide at the PAM-distal end of the region, the y-axis shows the position of the nucleotide position at the PAM-proximal end, and the z-axis show the value of r. Positive values are shown in brick red and negative values in sky blue. The correlation coefficient is at the highest between mutagenesis efficiency and the GC content of the six nucleotides closest to the PAM. See also Figure S2.

Figure 3

Figure 3. Triple and quadruple mutations in Drosophila with one shot

(A) Representative images of control flies, triple mutant F1s, and quadruple mutant F1s. The genotypes are listed on the top. (B) Triple and quadruple mutagenesis rates shown in a bar graph. Each row represents an sgRNA combination and the corresponding mutagenesis rate. Low GC content sgRNAs have less than 3 GCs in the 6 PAM-proximal nucleotides (PAMPNs), while high GC content ones have 5 or 6. See also Table S5.

Figure 4

Figure 4. Mutating HP1a through HDR using optimal sgRNAs

(A) Diagrams showing the repair donor plasmid and the mutation after HDR. The coding sequence of the HP1a locus is illustrated by the white boxes, and the 5’ and 3’ UTRs by the shaded boxes. The HP1a donor _HP1a_-4XP3-mCherry contains a 4XP3-mCherry sequence (red box) to replace most of the coding sequence of HP1a. The two homologous arms (HA-L and HA-R; blue) of the donor template are 0.97k bp and 1.2k bp, respectively. The Cas9/sgRNA cutting sites are denoted by the scissors. Successful replacement can be detected by mCherry expression in the fly eyes, or by PCR to detect the left and the right homologous arms. (B) Images of w1118 control and successful HP1aHDR-mCherry heterozygous mutant flies under bright-field (top) or epifluorescent light sources to show mCherry expression in the eyes (bottom). (C) Agarose gel electrophoresis with PCR band sizes confirming the successful HP1a HDR mutation. See also Figure S3.

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