Abstract
A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants. Guided by single guide (sg)RNAs, dCas9-AID-P182X (AIDx) directly changed cytidines or guanines to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9-AIDx converted targeted cytidines specifically to thymines, creating specific point mutations. By targeting BCR-ABL with dCas9-AIDx, we efficiently identified known and new mutations conferring imatinib resistance in chronic myeloid leukemia cells. Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution.
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Acknowledgements
We thank B. Li and C. Jiang for suggestions on data analysis; H. Zhang and A. Chen for assistance with high-throughput sequencing. This work was supported by 31370858 from National Natural Science Foundation of China (NSFC), 2014CB943600 from Ministry of Science and Technology (China) (MOST), 13PJ1409300 from Shanghai Municipal Science and Technology Committee (SMSTC), and National Thousand Talents Program for Distinguished Young Scholars (China).
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Y.M. and J.Z. performed experiments, analyzed data and wrote the manuscript. W.Y., Z.Z. and Y.S. assisted with the preparation of reagents, sequencing, data analysis and manuscript preparation. X.C. conceptualized the project, designed and supervised the research, and wrote the manuscript.
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X.C. has filed intellectual protection of using dCas9-AIDx for genetic diversification and protein evolution (application no. CN201610423512.8).
Integrated supplementary information
Supplementary Figure 1 dCas9-AIDx required enzymatic activity of AID for correcting a premature stop codon in GFP.
a. GFP sequence with a premature stop codon. The premature stop codon is labeled in red with yellow highlight. sgRNAs (indicated by arrows) and PAM sequences (in blue) are specified.
b. dCas9-AIDx requires enzymatic activity of AID. sgRNAs targeting GFP were co-transfected with Cas9, dCas9, dCas9-AID-P182X (E58Q) or dCas9-AID-P182X. Seven days after transfection, the cells were analyzed for GFP expression by flow cytometry.
Supplementary Figure 2 dCas9-AIDx efficiently creates mutations at the AAVS1 locus.
a. Design and sequences of sgRNAs targeting the AAVS1 locus (Hg19, chr19: 55626975- 55627279).
b-e. As in Fig. 2, dCas9-AIDx was transfected into 293T cells with three sgRNAs targeting the AAVS1 locus (e). Seven days after transfection, AAVS1 locus was PCR amplified and subjected to Miseq sequencing and mutations in the AAVS1 locus was analyzed. Substitution frequencies were calculated as reads with substitutions/ total reads covering the indicated bases (%). Parental 293T cells (b), cells transfected with AIDx(c), and cells transfected with dCas9-AIDx together with sgRNAs against GFP were used as control(d). sgRNA targeted regions were marked with gray boxes, and arrows indicate the location and direction of the sgRNAs-targeted DNA. Note, the y-axis is in log scale. Data are representative of three independent experiments.
Note, due to the lack of AID hotspot motifs in the AAVS1 locus, AIDx itself failed to induce mutations above the background.
Supplementary Figure 3 dCas9-AIDx efficiently diversifies sgRNA-targeted DNA.
The reporter cells were transfected with dCas9-AIDx together with either sgRNAs targeting GFP or sgRNAs targeting AAVS1. DNA sequences being 250-400bp downstream of the start codon of GFP (GFP) or within a 100 bp region of the AAVS1 locus (AAVS1) were considered as sgRNA-targeted regions respectively for the analysis.
a. The percentages of C /G nucleotides being mutated (substitution frequency >=0.1%) out of total C/ G were summarized from three independent experiments.
b. Average mutation rates (per base per cell cycle) of the C/G nucleotides in the sgRNA-targeted GFP or AAVS1 DNA were determined. Data are the summary of three independent experiments, and error bars show the standard deviation of the mean. **, p<0.01 in two-tailed Student’s t test.
c. The number of mutation combinations containing one or two mutant bases within the targeted region (GFP, 250 to 400; AAVS1, 201 to 300) was determined in 293T cells transfected with dCas9-AIDx and pooled sgRNAs targeting GFP or AAVS1. Data are the summary of two independent experiments, and error bars show the range. *, p<0.05 in two-tailed Student’s t test.
Supplementary Figure 4 Synergistic effects were observed by multiple sgRNAs targeting the same DNA window in inducing SNVs.
a. WT Cas9 but not dCas9-AIDx induces indels. The reporter cells were transfected with pooled sgRNA against GFP together with WT Cas9 or dCas9-AIDx. The indels in the GFP DNA were analyzed by CRISPResso. Data are summary of three independent experiments. Error bars stand for the standard deviation of the mean.
b,c. Synergistic effects of multiple sgRNAs targeting the same DNA. The reporter cells were transfected with dCas9-AIDx together with the pool of four sgRNAs(b) or one sgRNA(c). The substitution frequencies were calculated as reads with substitutions/total reads covering the base (%), and sgRNA targeted regions are shaded in gray. The x-axis indicates the relative position (bp) to the start codon of the GFP DNA. Note, the scale of y-axis in b (pooled sgRNAs) is 10-20 times greater than it in c (single sgRNA). Data are representative of three independent experiments.
d. dCas9-AIDx converts C or G into the other three bases. Proportions of each substitution out of total substitution for each nucleotide (% of total substitution) were summarized from three independent experiments. Error bars stand for the standard deviation of the mean. **, p<0.01; *, p<0.05 in two tailed Student’s t test.
Supplementary Figure 5 dCas9-AIDx did not induce nucleotide substitution in AID off-target loci.
As in Figure 2, the reporter cells were transfected with AIDx (a,c,e) or dCas9-AIDx together pooled-sgRNAs against GFP or AAVS1 (b,d,f), and sequence variants in the c-Myc (a,b Hg19 chr8: 128748974 -128749156), Bcl6 (c,d Hg19 chr3: 187462766 - 187463044) or PIM1 (e,f Hg19 chr6: 37138381- 37138554) were determined by sequencing. Note AIDx, but not dCas9-AIDx itself introduced a few mutations in these sites. Data are representative of three independent experiments.
g,h. The reporter cells were transfected with AIDx or dCas9-AIDx with pooled sgRNAs against AAVS1, and mutations in the GFP DNA were analyzed. Data are representative of three independent experiments.
Supplementary Figure 6 dCas9-AIDx induced mutagenesis at sgRNA off-target sites.
As in Fig. 2, HEK293T cells were transfected with dCas9-AIDx and pooled sgRNAs against GFP (a) or against AAVS1 (b). The off-target sites of indicated sgRNAs were predicted as described52, and top three off-target sites of each sgRNAs were amplified and sequenced. The mutation frequencies of nucleosides in the protospacer were determined. The mismatched and mutated bases were labeled with blue or red respectively. Data shown are average of two independent experiments.
Supplementary Figure 7 dCas9-AIDx preferentially mutated cytidines in protospacers when paired with a single sgRNA.
HEK293T reporter cells were transfected with dCas9-AIDx and Ugi expression constructs, together with individual sgRNA against GFP (a) or individual sgRNA against AAVS1 (b). Seven days after transfection, amplified DNA from GFP or AAVS1 locus was sequenced and substitution frequency was calculated as reads with substitutions/total reads covering the base (%). sgRNA targeted sequences were highlighted with gray boxes and marked with arrows for direction. Four nucleotides with the highest substitution frequencies were indicated on the sequence with grey boxes. The gradient of gray indicates relative substitution frequencies. The most frequent substitution with each individual sgRNA is also indicated. Data are representative of two independent experiments.
Supplementary Figure 8 Blocking UNG reveals the footprint of dCas9-AIDx on DNA.
a. dCas9-AIDx induces nucleotide substitutions predominantly within protospacer with highest activity at -12 and -16 bps upstream of the PAM sequence. 293T cells were transfected with dCas9-AIDx, Ugi and a single sgRNA, and substitution frequency was calculated as reads with C to T substitution/ total reads covering the base (%) within a -20bp to + 50 bp window relative to the PAM sequence. The x-axis indicates the relative location to PAM sequence (bp). The aggregated substitution frequency is denoted on the y-axis based on 12 individual sgRNAs (4 against GFP, 3 against AAVS1 and 5 against ABL kinase). Data are representative of two independent experiments.
b. dCas9-AIDx has progressive activity beyond protospacer when combined with multiple sgRNAs targeting the same region of DNA. The reporter cells were transfected with dCas9-AIDx, Ugi and pooled sgRNAs against GFP as in Fig. 2. Substitution frequency was calculated as reads with substitutions/total reads covering the base (%). Note, almost all the C/G nucleotides were mutated (substitution frequency >0.1%, dash line in the figure) from the 5’ of the first sgRNA to the 3’ end of the fourth sgRNA. Data are representative of two independent experiments.
c. as in b, the reporter cells were transfected with dCas9-AIDx, Ugi and pooled sgRNAs against AAVS1. The substitution frequency of the target DNA was determined as above. Data are representative of two independent experiments.
Supplementary Figure 9 Design and sequences of sgRNAs targeting exon 6 of ABL kinase.
(a) Domain structure of ABL kinase; (b). sgRNAs were designed to target Exon 6 ±60 bps upstream and downstream. Exon sequence is capitalized and marked in gray. (c). cDNA of exon 6 in ABL gene was amplified and sequenced from DMSO treated K562 cells. The substitution frequency of each base was calculated as reads with substitutions/ total reads covering the base (%).
Supplementary Figure 10 Abl mutations identified in the TAM screening confer Imatinib resistance in K562 cells.
(a). Summary of the point mutations identified in two independent Imatinib-resistance screenings. The nucleotide and amino acid substitution of each mutation are presented together with their substitution frequencies. The mutations identified in both experiments were highlighted with yellow. Note C956 were mutated to either G or T in the first screening, which leads to T319S or T319I mutation respectively.
(b,c) The indicated ABL mutations were introduced into an MSCV-BCR/ABL-IRES-GFP vector, which was used to transduce K562 cells. The GFP+ cells were sorted and treated with either 4μM (b) or 10μM (c) Imatinib. The cells were counted everyday after the Imatinib treatment. Data are representative of two independent experiments.
Supplementary Figure 11 Cas9-nickase (nCas9)-AIDx enhanced the mutagenesis efficiency with single sgRNA but caused extensive indels with pooled sgRNAs.
a. nCas9 with AIDx enhanced nucleoside conversion efficiency. Reporter cells were transfected with a single sgRNA against GFP together with either dCas9-AIDx+Ugi (left) or nCas9-AIDx+Ugi (right). Substitution frequencies of each base were calculated.
b,c. nCas9-AIDx failed to turn on GFP. The reporter cells were transfected with nCas9-AIDx (right) together with AAVS1 sgRNA (Ctrl sgRNA) or pooled GFP sgRNAs. GFP expression was determined seven days after transfection. dCas9-AIDx (left) was shown as positive controls. b, Representative FACS plots; c, summary of three independent experiments.
d. nCas9-AIDx induced extensive Indels when combined with pooled sgRNAs against GFP. As in b, Indels in the GFP locus were determined by high-throughput sequencing. Summary of two independent experiments. Error bars stand for the standard deviation of the mean. **, p<0.01 in two tailed Student’s t test.
Supplementary Figure 12 Saturation analysis for the number of sgRNAs required in a TAM screening.
293T cells were transfected with dCas9-AIDx together with 10 sgRNAs (a), 7 sgRNAs (b), 5 sgRNAs (c) or 3 sgRNAs (d) targeting a 100bp window (201-300bp) within the exon 6 of ABL kinase. In the case of 10 sgRNAs, 3 sgRNAs targeting adjacent to this region are included, since only 7 sgRNAs can be designed within the 201-300 bp window. Substitution frequencies were calculated as reads with substitutions/ total reads covering the indicated bases (%). sgRNA targeted regions were marked with gray boxes, and arrows indicate the location and direction of the sgRNAs-targeted DNA. Data are representative of two independent experiments.
(e) As in Fig. S3a, percentages of mutated C/G nucleosides were calculated and compared among cells receiving indicated number of sgRNAs.
(f), substitution frequencies of mutant C/Gs were plotted and compared ompared among cells receiving indicated number of sgRNAs.
(g) As in Fig.S3c the number of mutation combinations within the targeted region (201 to 300) was determined in 293T cells transfected with dCas9-AIDx and indicated number of pooled sgRNAs targeting the ABL kinase.
Data are summary of two independent experiments. Error bars stand for the standard deviation of the mean. *, p<0.05 in two tailed Student’s t test.
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Ma, Y., Zhang, J., Yin, W. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat Methods 13, 1029–1035 (2016). https://doi.org/10.1038/nmeth.4027
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DOI: https://doi.org/10.1038/nmeth.4027
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