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, 55 (40), 12440-4

Development of Light-Activated CRISPR Using Guide RNAs With Photocleavable Protectors

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Development of Light-Activated CRISPR Using Guide RNAs With Photocleavable Protectors

Piyush K Jain et al. Angew Chem Int Ed Engl.

Abstract

The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. In this work, we have developed a method to photocage the activity of the guide RNA called "CRISPR-plus" (CRISPR-precise light-mediated unveiling of sgRNAs). The photoactivation capability of our CRISPR-plus method is compatible with the simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic investigations.

Keywords: CRISPR; DNA cleavage; gene technology; nucleic acids; photochemistry.

Figures

Figure 1
Figure 1
CRISPR-plus achieves photoactivatable blockade of Cas9-mediated DNA targeting. (a) CRISPR-plus concept. (b) Fold increase in DNA cleavage following light exposure (365 nm, 6.0 J/cm2) with complementary sgGFP1 protectors of different lengths and position that contain photocleavable (PC) groups spaced 6 nucleotides apart (PCT1–PCT5), or protectors without PC groups (T1–T5), tested using an in vitro cleavage assay (n=2) (c) Representative gel electrophoresis data from the in vitro cleavage assay, for a 2:1 ratio of protector PCT5:sgRNA. The 702 bp target GFP DNA is cut to yield 563 bp and 139 bp fragments in the presence of sgRNA, only after p-sgRNA photolysis (365 nm, 6.0 J/cm2). (d) Quantification of replicate in vitro assays from (c) (n=3), where data is expressed as a fraction of uncleaved DNA, as calculated based on band intensities. Data is normalized to control untreated DNA, and mean values with standard deviation are plotted (n=3). Unpaired student t-test was performed for sgRNA with PCT5 in the presence or absence of light exposure groups and p values are represented with asterisks ***p < 0.001.
Figure 2
Figure 2
Validation of optimized 24-nt target-specific protectors with other sgRNAs and targets in an in vitro DNA cleavage assay. (a–c) PC-containing (PCT6–PCT11) or non-PC (T6–T11) oligonucleotides complementary to 6 additional sgRNAs targeting three different DNA targets (a) GFP (green), (b) CD71 (magenta), and (c) CD33 (blue), in the absence or presence of light (365 nm, 6.0 J/cm2). % uncleaved DNA was calculated from the band intensities of the gels (SI Figure S6). Data is normalized to the cleavage of control, untreated DNA. Mean values +/− s.d. are plotted for multiple repeat experiments (sgGFP-2, sgGFP-3 and sgCD71-1, n=4; sgCD71-2 and sgCD33-1, n=2; sgCD33-3, n=2 with T11, n=1). Unpaired student’s t-test was performed between irradiated and non-irradiated samples, as described in the SI data analysis section, and p values are represented with asterisks *p < 0.05, **p < 0.01, ****p < 0.0001.
Figure 3
Figure 3
CRISPR-plus enables simultaneous targeting of multiple genes. (a) Schematic representation of combining three different sgRNAs with respective protectors and their activation with light. (b) Simultaneous in vitro targeting of three genes (GFP, CD71 and CD33) by their corresponding sgRNAs (sgGFP (green), sgCD71 (magenta), and sgCD33 (blue)) with (p-sgRNA) or without (sgRNA) their cognate photolabile protectors in the absence (solid bars) or presence (open bars) of light (365 nm, 6.0 J/cm2). The proportion of uncleaved DNA is expressed relative to untargeted DNA, averaged over two individual experiments and sgCon bars represent non-targeting scrambled sgGFP-1. Error bars represent standard deviation (n=2). DNA cleavage was blocked only in the presence of the corresponding protector, and all the p-sgRNA-treated conditions regained CRISPR activity in the presence of light.
Figure 4
Figure 4
Validation of CRISPR-plus in cells. (a, b) SURVEYOR nuclease assay was performed 72 hours post-transfection using Cas9/d2eGFP-expressing HeLa cells transfected with sgGFP-1 (a), or sgCD71-1 (b), with PCT5 or PCT8, respectively, in the absence or presence of light. Uncleaved GFP DNA (green, 500 bp) cut to a shorter fragment (black, 408 bp), and uncleaved CD71 DNA (magenta, 434 bp) with its 350 bp & 84 bp cleavage fragments are indicated. (c) Representative flow cytometry histograms of Cas9/d2eGFP-expressing HeLa cells transfected with sgGFP-1, with or without PCT5. The presence or absence of light (365 nm, 4.0 J/cm2) is indicated for each condition and control traces (top) represent untreated cells. Additional controls included in SI Figure S9. (d) Quantification of the fraction of GFP negative cells observed in (c), where mean values are plotted with error bars indicating standard deviation (n=3). Unpaired student’s t-test was performed for sgRNA with PCT5 in the presence or absence of light exposure groups and p values are represented with asterisks ***p < 0.001.

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