Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations
Summary (3 min read)
Introduction
- To recognize its sequence, Cas9 requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene.
- In addition, the authors analyzed off-target mutations genomewide and found that three microsatellites with similar sequences were also edited by the nuclease.
Yeast plasmids
- A synthetic cassette was ordered from ThermoFisher .
- The I-Sce I site was flanked by Sap I recognition sequences, in order to clone the different repeat tracts.
- Each not certified by peer review) is the author/funder.
- Nucleases were amplified from Addgene plasmids indicated in Supplemental Table S1.
- SaCas9 and FnCpf1 guide RNAs were ordered at Twist Biosciences, directly cloned into pRS416 (see Supplemental Table S1 for plasmid names).
Yeast strains
- Each synYEGFP cassette containing repeat tracts was digested by Bam HI in order to linearize it and transformed into the FYBL1-4D strain (Gietz et al., 1995).
- Correct integrations at the CAN1 locus were first screened as [CanR, Trp+] transformants, on SC - ARG -TRP +Canavanine (60 μ/ml) plates.
- Repeats were amplified by PCR using LP30bLP33b primers and sequenced (Eurofins/GATC).
- As a final confirmation, all transformants were also analyzed by Southern blot and all the [CanR, Trp+] clones showed the expected profile at the CAN1 locus.
- Derived strains were called LPY101 to LPY111 (Supplemental Table S3).
Flow cytometry assay
- Cells were transformed using standard lithium-acetate protocol (Gietz et al., 1995) with both guide and nuclease and selected on 2% glucose SC -URA -LEU plates and grown for 36 not certified by peer review) is the author/funder.
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- Each colony was then picked and seeded into a 96-well plate containing 300 μL of either 2% glucose SC -URA -LEU or 2% galactose SC -URA -LEU.
- At each time point (0h, 12h, 24h, 36h) cells were diluted in PBS and quantified by flow cytometry after gating on homogenous population, single cells and GFP-positive cells.
- The complete protocol was extensively described in (Poggi et al., 2020).
Southern blot analyses
- For each Southern blot, 3-5 μg of genomic DNA digested with Eco RV and Ssp I were loaded on a 1% agarose gel and electrophoresis was performed overnight at 1V/cm.
- The gel was manually transferred overnight in 20X SSC, on a Hybond-XL nylon membrane (GE Healthcare), according to manufacturer recommendations.
- Hybridization was performed with a 32P-randomly labeled CAN1 probe amplified from primers CAN133 and CAN135 (Supplemental Table S2) (Viterbo et al., 2018).
- The membrane was exposed 3 days on a phosphor screen and quantifications were performed on a FujiFilm FLA-9000 phosphorimager, using the Multi Gauge (v. 3.0) software.
- Percentages of DSB and not certified by peer review) is the author/funder.
Agarose plug DNA preparation
- During time courses of DSB induction (see above), 2 x 109 cells were collected at each time point and centrifuged.
- This mix was rapidly poured into plug molds and left in the cold room for at least 10 minutes.
- TE was replaced by 1 mL restriction enzyme buffer (Invitrogen REACT 2) for one hour, then replaced by 100 μl buffer containing 100 units of each enzyme (Eco RV and Ssp I) and left overnight at 37°C.
- The liquid phase not certified by peer review) is the author/funder.
Northern blot analyses
- Each repeat-containing strain transformed with its cognate gRNA and nucleases was grown for 4 hours in 2% galactose SC-URA-LEU.
- Total RNAs were extracted using standard phenol-chloroform procedure (Richard et al., 1997) or the miRVANA kit, used to extract very low levels of small RNAs with high efficacy .
- Total RNA samples were loaded on 50% urea 10% polyacrylamide gels and run at 20 W for one hour.
- Gels were electroblotted on N+ nylon membranes (GE Healthcare), hybridized at 42°C using a SpCas9, SaCas9, FnCpf1 or SNR44 oligonucleotidic probe.
- Each probe was terminally labeled with γ32P ATP in the presence of polynucleotide kinase.
Western blot analyses
- Total proteins were extracted in 2X Laemmli buffer and denatured at 95°C before being loaded on a 12% polyacrylamide gel.
- Membranes were washed in TBS-T for 10 minutes twice.
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- Ratios of relative resection rates from both sides of the repeated sequence were calculated and compared to a non-repeated control sequence.
Determination of off-target mutations
- Cell were grown overnight in YPGal medium and diluted for 2 more hours.
- The copyright holder for this preprint (which wasthis version posted November 29, 2019.
- Regarding reads that did not contain the dsODN tag, mutations within predicted off-target sites were detected by the mean of samtools pileup applied to all regions of interest identified by crispor (Haeussler et al., 2016).
Statistical Analysis
- All statistical tests were performed with R3.5.1.
- Linear regression was performed to determine statistical significance of proteins levels and gRNA levels over the percentage of GFP-positive cells.
- For each linear regression, R2 and p-value were calculated.
- P-values less than 0.05 were considered significant.
- Figures were plotted using the package ggplot2.
Results
- A GFP reporter assay integrated in the Saccharomyces cerevisiae genome enables the quantification of nuclease activity.
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- CTG repeats and CAG repeats were not cut the same way, eSpCas9 being more efficient on (CAG)33 than SpCas9, although the contrary was found for (CTG)33 .
- No correlation was found between gRNA quantification and GFP-positive cells, showing that gRNA steady state level was not the limiting factor in this reaction .
- BioRxiv preprint 22 whether these mutant reads were statistically significant, they were compared to the number of mutant reads at the same positions in the NR library used as a control.
Discussion
- Here, the authors successfully designed an assay for determining Cas9 variant efficacy on various microsatellites.
- The copyright holder for this preprint (which wasthis version posted November 29, 2019.
- BioRxiv preprint 23 Previous biophysical analyses showed that Cas9-HF1 and eSpCas9 bound to DNA similarly to SpCas9, but variants were trapped in an inactive state when bound to off-target sequences (Chen et al., 2017).
- In their assay, replication may also convert a nick into a DSB, triggering homologous recombination in repeated sequences as suggested by the presence of a DSB observed throughout repair time course .
- Reducing the expression period of the nuclease should also help reducing off-target mutations, but this has now to be thoughtfully investigated.
Acknowlegments
- Off-target studies were supported by the AFM-Telethon.
- The authors thank Heloïse Muller for sharing her unpublished protocol for yeast transformation by electroporation, and Carine Giovannangeli for the generous gift of CRISPR-Cas plasmids.
- This work was supported by Sanofi, the Institut Pasteur and the Centre National de la Recherche Scientifique (CNRS).
- Not certified by peer review) is the author/funder.
- The copyright holder for this preprint (which wasthis version posted November 29, 2019.
Figure legends
- The CAN1 locus was replaced by recombinant GFP cassettes, also known as A.
- Recombination efficacies are indicated by the same color code as in Figure 2A.
- BioRxiv preprint 34 Reconstructed models of SpCas9 (left) and eSpCas9 interacting with a structured CAG/CTG repeat, according to the SpCas9 crystal structure (PDB: 4UN3).
- GFP-positive cells as a function of Gibbs energy calculated for each gRNA alone, also known as C.
- Genome coverage was calculated by dividing not certified by peer review) is the author/funder.
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References
152 citations
"Differential efficacies of Cas nucl..." refers background in this paper
...This has been extensively studied and reviewed over the last 25 years (Gacy et al., 1995; Lenzmeier and Freudenreich, 2003; McMurray, 2010; Mirkin, 2006; Pearson et al., 2005; Richard et al., 2008; Usdin et al., 2015)....
[...]
148 citations
"Differential efficacies of Cas nucl..." refers methods in this paper
...Yeast strains Each synYEGFP cassette containing repeat tracts was digested by Bam HI in order to linearize it and transformed into the FYBL1-4D strain (Gietz et al., 1995)....
[...]
...Flow cytometry assay Cells were transformed using standard lithium-acetate protocol (Gietz et al., 1995) with both guide and nuclease and selected on 2% glucose SC -URA -LEU plates and grown for 36 hours....
[...]
...Time courses of DSB inductions Cells were transformed using standard lithium-acetate protocol (Gietz et al., 1995) with both guide and nuclease and selected on 2% glucose SC -URA -LEU plates and grown for 36 hours....
[...]
132 citations
"Differential efficacies of Cas nucl..." refers methods in this paper
...Later on, ZFNs were used to induce DSBs into CAG or CTG repeats, which mostly led to contractions in CHO cells (Mittelman et al., 2009) and in a HEK293 cell GFP reporter assay (Santillan et al., 2014)....
[...]
...Later on, ZFNs were used to induce DSBs into CAG or CTG repeats, which mostly led to contractions in CHO cells (Mittelman et al., 2009) and in a HEK293 cell GFP reporter assay (Santillan et al....
[...]
130 citations
"Differential efficacies of Cas nucl..." refers background in this paper
...By comparison, the human genome contains 900 or 1356 CAG/CTG repeats, depending on authors (Kozlowski et al., 2010)(Lander et al., 2001)....
[...]
...By comparison, the human genome contains 900 or 1356 CAG/CTG repeats, depending on authors (Kozlowski et al., 2010)(Lander et al....
[...]
124 citations
"Differential efficacies of Cas nucl..." refers background in this paper
...pombe, mating type switching occurs by homologous recombination after the conversion of a nick into a DSB during replication (Arcangioli, 1998; Dalgaard and Klar, 2001)....
[...]
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Frequently Asked Questions (2)
Q2. What are the future works mentioned in the paper "Differential efficacies of cas nucleases on microsatellites involved in human disorders and associated off-target mutations" ?
However, their results allow to discard inefficient nucleases for further human studies. This suggests that random breakage occurs frequently within these repeated sequences.