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Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations

29 Nov 2019-bioRxiv (Cold Spring Harbor Laboratory)-pp 857714

TL;DR: It is demonstrated that secondary structure formation on the guide RNA was a major determinant of nuclease efficacy, and the most efficient of all CRISPR-Cas nucleases was Streptococcus pyogenes Cas9.

AbstractMicrosatellite expansions are the cause of more than 20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, a new assay was developed to test several CRISPR-Cas nucleases on microsatellites involved in human diseases, by measuring at the same time double-strand break rates, DNA end resection and homologous recombination efficacy. Broad variations in nuclease performances were detected on all repeat tracts. Streptococcus pyogenes Cas9 was the most efficient of all. All repeat tracts did inhibit double-strand break resection. We demonstrate that secondary structure formation on the guide RNA was a major determinant of nuclease efficacy. Using deep sequencing, off-target mutations were assessed genomewide. Out of 221 CAG/CTG or GAA/TTC trinucleotide repeats of the yeast genome, three were identified as carrying statistically significant low frequency mutations, corresponding to off-target effects.

Topics: Cas9 (53%), Microsatellite (52%), Homologous recombination (52%), Gene (52%), DNA (51%)

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  • Each not certified by peer review) is the author/funder.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.
  • The copyright holder for this preprint (which wasthis version posted November 29, 2019.
  • 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.
  • The copyright holder for this preprint (which wasthis version posted November 29, 2019.
  • 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.
  • The copyright holder for this preprint (which wasthis version posted November 29, 2019.
  • 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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1
Differential efficacies of Cas nucleases on microsatellites involved in human disorders
and associated off-target mutations
Lucie Poggi
1,2,3
, Lisa Emmenegger
1,4
, Stéphane Descorps-Declère
1,5
, Bruno Dumas
3
, Guy-
Franck Richard
1,2
1 Institut Pasteur, CNRS, UMR3525, 25 rue du Dr Roux, F-75015 Paris, France
2 Sorbonne Université, Collège Doctoral, 4 Place Jussieu, F-75005 Paris, France
3 Biologics Research, Sanofi R&D, 13 Quai Jules Guesde, 94403 Vitry sur Seine, France
4 Present address: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for
Molecular Medicine in the Helmholtz Association, Robert-Rössle-Strasse 10, 13125 Berlin,
Germany.
5 Institut Pasteur, Bioinformatics and Biostatistics Hub, Department of Computational
Biology, USR3756 CNRS, F-75015 Paris, France
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint

2
Abstract
Microsatellite expansions are the cause of more than 20 neurological or developmental human
disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned
as a gene editing approach. Here, a new assay was developed to test several CRISPR-Cas
nucleases on microsatellites involved in human diseases, by measuring at the same time
double-strand break rates, DNA end resection and homologous recombination efficacy. Broad
variations in nuclease performances were detected on all repeat tracts.
Streptococcus pyogenes Cas9 was the most efficient of all. All repeat tracts did inhibit
double-strand break resection. We demonstrate that secondary structure formation on the
guide RNA was a major determinant of nuclease efficacy. Using deep sequencing, off-target
mutations were assessed genomewide. Out of 221 CAG/CTG or GAA/TTC trinucleotide
repeats of the yeast genome, three were identified as carrying statistically significant low
frequency mutations, corresponding to off-target effects.
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint

3
Introduction
A growing number of neurological disorders were identified to be linked to microsatellite
expansions (Orr and Zoghbi, 2007). Each disease is associated to a repeat expansion at a
specific locus (Table 1). No cure exists for any of these dramatic disorders. Shortening the
expanded array to non-pathological length could suppress symptoms of the pathology and
could be used as a new gene therapy approach (Richard, 2015). Indeed, when a trinucleotide
repeat contraction occurred during transmission from father to daughter of an expanded
myotonic dystrophy type 1 allele, clinical examination of the daughter showed no sign of the
disease (O’Hoy et al., 1993) (Shelbourne et al., 1992).
In order to induce a double-strand break (DSB) into a microsatellite, different types of
nucleases can be used: meganucleases, Zinc Finger Nucleases (ZFN), Transcription activator-
like effector nucleases (TALEN) and CRISPR-Cas9. Previous experiments using the I-SceI
meganuclease to induce a DSB into a CTG repeat tract showed that repair occurred by
annealing between the flanking CTG repeats (Richard et al., 1999). 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). As
only one arm was enough to induce a DSB into the repeat tract and since CAG zinc fingers
can recognize CTG triplets and vice versa, the authors concluded that the specificity was too
low for further medical applications. As a proof of concept of the approach, a myotonic
dystrophy type 1 CTG repeat expansion was integrated into a yeast strain. A TALEN was
designed to recognize and cut the CTG triplet repeat and was very efficient at shortening it in
yeast cells (>99% cells showed contraction) and highly specific as no other mutation was
detected (Richard et al., 2014). The TALEN was shown to induce specific repeat contractions
through single-strand annealing (SSA) by a RAD52, RAD50 and SAE2 dependent mechanism
(Mosbach et al., 2018). As a proof of concept of the approach, a myotonic dystrophy type 1
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint

4
CTG repeat expansion was integrated into a yeast strain. A TALEN was designed to
recognize and cut the CTG triplet repeat and was very efficient at shortening it in yeast cells
(>99% cells showed contraction) and highly specific as no other mutation was detected
(Richard et al., 2014).
The CRISPR-Cas system is the easiest to manipulate and to target any locus, as sequence
recognition is based on the complementarity to a guide RNA (gRNA). To recognize its
sequence, Cas9 requires a specific protospacer adjacent motif (PAM) that varies depending on
the bacterial species of the Cas9 gene. The most widely used Cas9 is wild-type Streptococcus
pyogenes Cas9 (SpCas9) (Cong et al., 2013). Its Protospacer Adjacent Motif (PAM) is NGG
and induces a blunt cut 3-4 nucleotides away from it, through concerted activation of two
catalytical domains, RuvC and HNH, each catalyzing one single-strand break (SSB). Issues
were recently raised about the specificity of SpCas9, leading to the engineering of more
specific variants. In eSpCas9, three positively charged residues interacting with the phosphate
backbone of the non-target strand were neutralized, conferring an increased specificity
(Kleinstiver et al., 2016). Similarly, Cas9-HF1 was mutated on 4 residues interacting through
hydrogen bonds with the target strand (Slaymaker et al., 2016). Staphylococcus aureus is a
smaller Cas9, its PAM is NNGRRT, having a similar structure to SpCas9 with two catalytic
sites. Finally, type V CRISPR-Cas, Cpf1 nucleases, exhibit very different features including a
T-rich PAM located 3’ of target DNA and making staggered cuts leaving five-nucleotide
overhangs by iterative activation of a single RuvC catalytic site (Zetsche et al., 2015).
Cutting repeated sequences like microsatellites may be difficult due to stable secondary
structures that may form either on target DNA or on the guide RNA, making some repeats
more or less permissive to nuclease recognition and cleavage. In addition, secondary structure
formation could impede DSB resection or later repair steps. Eukaryotic genomes contain
thousands of identical microsatellites, therefore the specificity issue may become a real
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint

5
problem when targeting one single locus. Here we developed an in vivo assay in the yeast
Saccharomyces cerevisiae in order to test different nucleases belonging to the CRISPR-Cas
family on synthetic microsatellites associated to human disorders. Our experiments revealed
that these sequences may be cut, with surprisingly different efficacies between nucleases and
between microsatellites. SpCas9 was the most efficient and nuclease efficacy relied mainly on
gRNA stability, strongly suggesting that secondary structures are the limiting factor in
inducing a DSB in vivo. DSB resection was decreased to different levels in all repeated tracts.
In addition, we analyzed off-target mutations genomewide and found that three microsatellites
with similar sequences were also edited by the nuclease. The mutation pattern was different
depending on the microsatellite targeted.
Materials and Methods
Yeast plasmids
A synthetic cassette (synYEGFP) was ordered from ThermoFisher (GeneArt). It is a pUC57
vector containing upstream and downstream CAN1 homology sequences flanking a bipartite
eGFP gene interrupted by the I-Sce I recognition sequence (18 bp) under the control of the
TEF1 promoter and followed by the CYC1 terminator. The TRP1 selection marker along with
its own promoter and terminator regions was added downstream the eGFP sequences (Figure
1A). The I-Sce I site was flanked by Sap I recognition sequences, in order to clone the
different repeat tracts. Nine out the 10 repeat tracts were ordered from ThermoFisher
(GeneArt) as 151 bp DNA fragments containing 100 bp of repeated sequence flanked by Sap
I sites. The last repeat (GGGGCC) was ordered from Proteogenix. All these repeat tracts were
cloned at the Sap I site of synYEGFP by standard procedures, to give plasmids pLPX101 to
pLPX110 (Supplemental Table S1). All nucleases were cloned in a centromeric yeast plasmid
derived from pRS415 (Sikorski and Hieter, 1989), carrying a LEU2 selection marker. Each
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 29, 2019. ; https://doi.org/10.1101/857714doi: bioRxiv preprint

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Frequently Asked Questions (2)
Q1. What are the contributions in "Differential efficacies of cas nucleases on microsatellites involved in human disorders and associated off-target mutations" ?

In this paper, the authors used a single-strand-annealing ( SSA ) mechanism to induce a double-strands break ( DSB ) in a CTG repeat tract. 

However, their results allow to discard inefficient nucleases for further human studies. This suggests that random breakage occurs frequently within these repeated sequences.