Materials and Methods

Role of nucleotide identity in effective CRISPR target escape mutations

MATERIALS AND METHODSBacterial strains and growth conditions Escherichia coli strain KD263 was obtained from (30). Escherichia coli strains were grown at 37°C in Luria Broth (LB; 5 g/l NaCl, 5 g/l yeast extract, and 10 g/l tryptone) at 180 rpm or on LB-agar plates containing 1.5% (w/v) agar. When required, medium was supplemented with the following: ampicillin (Amp; 100 μg/ml), chloramphenicol (Cm; 34 μg/ml), or kanamycin (Km; 50 μg/ml). Bacterial growth was measured at 600 nm (OD600).Molecular biology and DNA sequencingAll oligonucleotides are listed in Supplementary Table S3. All plasmids are listed in Supplementary Table S4. All strains and plasmids were confirmed by PCR and sequencing (GATC-Biotech). Plasmids were prepared using GeneJET Plasmid Miniprep Kits (Thermo Scientific). DNA from PCR was purified using the DNA Clean and Concentrator and Gel DNA Recovery Kit (Zymo Research). The protospacer plasmid set was constructed by cutting pWUR925 with XbaI and SacI, removing the kanamycin resistance marker, and ligating a PCR product containing the streptomycin resistance marker and the desired protospacer (primers: BG7167/7395-7 for controls, BG7167/8393-8410 for mutant set).Plasmid loss assayThe assay was carried out in E. coli KD263 cells, which have inducible cas gene expression. Expression was induced with 0.2% l-arabinose and 0.5 mM IPTG where appropriate. Escherichia coli KD263 cells were transformed with the target plasmids (pWUR926-946) by heat shock. Individual colonies were picked in duplicate and grown overnight in 5 ml LB supplemented with 2% glucose to repress cas gene expression. The next day, cultures were transferred 1:100 into induced medium (0.2% L-Arabinose, 0.5 mM IPTG) and plasmid loss was monitored. Samples were taken at the time of induction and 1.5, 3, 4.5, 6, 7, 24 and 48 h post induction (HPI). Dilutions were plated on non-selective plates containing 0.2% rhamnose and plasmid loss was quantified based on loss of red color. Liquid culture samples were screened for spacer integration by colony PCR using OneTaq (NEB). Acquisition of spacers was detected by PCR using primers BG5301 and BG5302. PCR products were visualized on 2% agarose gels and stained with SYBR-safe (Invitrogen). PCR products were sequenced using Sanger sequencing at GATC (Konstantz, Germany) using primer BG5301.Protein purificationAll proteins were expressed in BL21-AI cells. Cascade was purified as described earlier (31). MBP-Cas3 was purified as described in (32).Oligo annealing and labellingComplementary oligo nucleotides (BG9069-9074) were mixed (1:1) in a Tris-sodium buffer, heated to 95°C and slowly cooled to room temperature. Duplexes were checked on a native 20% acrylamide gel for residual single stranded oligo. The non-target substrate was PCR amplified from pWUR928 using BG9141/2. Duplexes were then labeled with γ-32P-ATP using T4 PNK (NEB) and free label was removed using a G25 column.EMSA assaysPurified Cascade complex with spacer8 crRNA was incubated with plasmid or oligos at a range of molar ratios (1:1-96:1, Cascade:DNA) in buffer A (20 mM HEPES pH 7.5, 75 mM NaCl, 1 mM DTT) for 30 min (33). Plasmid reactions were run on 1% native agarose gels for 18 h at 22 mA in 8 mM sodium-borate buffer. Gels were post stained with SYBR Safe (Invitrogen). Oligo reactions were run on 5% native acrylamide gels at 4 mA for 18 h. Gels were exposed to a phosphor screen (GE Healthcare) and scanned using a phosphor imager (Bio-Rad PMI). Shifted (Cascade bound DNA) and unshifted (free DNA) bands were quantified using the GeneTools software (Syngene) or ImageJ and free Cascade concentration (X) was plotted against the fraction of bound DNA (Y). The curves were fitted with the following formula: Y = (amplitude * X)/(Kd + X) (34). The amplitude is the maximum fraction of bound DNA. The affinity ratio is determined as amplitude/Kd to correct for the variable amplitudes (35).Cas3 DNA degradation assaysPlasmid-based assays were performed by incubating 70 nM Cas3 with 100 nM Cascade and 3.5 nM plasmid DNA. Reactions were incubated in buffer R (5 mM HEPES, pH 8, 60 mM KCl) supplemented with 10 μM CoCl2, 10 mM MgCl2, 2 mM ATP at 37°C for the indicated amount of time. Reactions were quenched on ice with 6× DNA loading dye (Thermo scientific). Reactions were run on 0.8% agarose gels at 100 V for 40 min and supercoiled plasmid bands were quantified using the GeneTools software (Syngene).Structural modeling of target mismatchesAtomic models of the Cascade complex bound to mismatched DNA targets were made with the molecular modeling program Coot (36). To visualize how G and C mismatches would affect target binding, the G and C mismatches of the C7/G7 target were modeled into the crRNA spacer sequence of dsDNA bound Cascade, (PDB: 5H9E), using the simple mutate tool in Coot. To model wobble basepairs the Rotate Translate Zone/Chain/Molecule tool was used to move nucleotides of the target strand as a rigid bodies into wobble positions. Rendering of atomic model images was performed with PyMOL Molecular Graphics System, Version 2.0 Schödinger, LLC.

Article TitleRole of nucleotide identity in effective CRISPR target escape mutations

Abstract

Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR interference and interference-dependent priming, while cytosine-substitutions are more readily tolerated. Furthermore, we show that this effect is based on strongly decreased binding affinity of the effector complex Cascade for guanine-mismatched targets, while cytosine-mismatched targets only minimally affect target DNA binding. Structural modeling of Cascade-bound targets with mismatches shows that steric clashes of mismatched guanines lead to unfavorable conformations of the RNA-DNA duplex. This effect has strong implications for the natural selection of target site mutations that lead to effective escape from type I CRISPR–Cas systems.


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