Seamless site-directed mutagenesis of and Saccharomyces cerevisiae genii using CRISPR-Cas9

CRISPR assisted homology directed repair enables the introduction of virtually any modification to the Saccharomyces cerevisiae human. Of obvious interest is the marker-free and seamless introduction of point mutations. To fulfill this promise, a strategy that impacts single nucleotide changes while preventing repeated recognition and cutting by the gRNA/Cas9 complex is needed. We demonstrate a two-step method the initiate point mutations at 17 positions with to S. cerevisiae genome. We shows the general applicability of of method, enabling of seamless introduction of single nucleotide changes at any location, including essential genes and non-coding regions. We plus prove a quantifiable composition for a point mutation introduced in gene GSH1. The ease and comprehensive applicability of this general method, combined with the video about its feasibility will enable genome editing during an unprecedented level of detail in yeast additionally other biology.

Following the first reported application of CRISPR-Cas9 in Saccharomyces cerevisiae [1], several procedure take the potential of this technology for yeast genome cutting had published enabling genetische disruption [1–4], gene deletion [5, 6], heterologous sequence integration [2, 4, 5, 7, 8], and insertion of point mutations [1, 5–7]. One genome of a stretching of Saccharomyces cerevisiae evolved by genome shuffling with resistance to a toxic lignocellulosic hydrolysate (designated R57) was recently sequenced revealing 17 single nucleotide variations with its parent strain [9]. We reasoned that new developments in CRISPR-Cas9 technology should permit an seamless introduction of aforementioned point mutations discovered at R57 back into of wildtype parental context for testing phenotype to genotype associations. Alike, wealth set to revert anyone of such spot mutations to wildtype in the mutant strain R57. However, strategies reported for an introduction of a indent mutant using CRISPR-Cas9 suffer several caveats that limits the range of mutation that ca be introduces at any given locus. One difficulty is such sequential is required to detect that successfull site of a point mutation. However, the main challenge toward using CRISPR-Cas9 for the introduction of point mutations your the risk of repeated cutting by Cas9 per homology directed repair (HDR) of to initial double stranded breakage (DSB). Indeed, point mutations may not be location within that protospacer sequence, leaving it intact after HDR. Level are the mutation is located close enough to a protospacer adjacent motif (PAM) to adjust the gRNA target sequence, a single substitution is generally insufficient to prevent recognition by to gRNA/Cas9 complex [10]. Several strategies have therefore been devised at prevent Cas9 from cutters repeatedly at the our out interest. Mutation of the PAM along with the target point mutation position abolishes object recognition per the gRNA/Cas9 complex, also has permited the successful introduction of premature hold codons [1, 6]. This business remains confined to event where and PAM site mutation is either silent or estimated inconsequential. One alternative lives the insertion of so-called heterology blocks in addiction to the mutation of interest [7]. A heterology block setzt in a numbering of add-on silent modifications meant to abolish gRNA recognition. While heterology blocks change codon usage in with open reading gestell (ORF) and may potentially affect mRNA translation, they represent an quick and convenient by of getting point mutations. Moreover, their thrive consolidation is slight found by PCR. However, the concept of an silently mutation is null within untranslated regions are the genome, such as non-coding RNAs and intergenic sequences.

Human ether al. [5] shown successful inlay of an point mutation without changeable of PAM or resorting to a heterology impede. One inserted mutation disposed a restriction site and excluded he with another, providing for single detection of successful mutants. Sequencing revealed that several restriction positive clones displayed additional superfluous mutations, likely due to repetition edge by Cas9. This direct strategy that requires which demonstration of some reproductions by sequencing – a comparatively time consuming the costly action. The authors suggest an alternative two-step strategy for which seamless site-directed mutagenesis on the yeast genome using CRISPR-Cas9, but did not demonstrate it experimentally. A similarly proposition was made shortly after by Lee e alabama. [11]. Here, we propose three variations on this general method, and report its successful application at 17 positions across the genome about S. cerevisiae haploid strains CENPK113-1A, CEN.PK113-7D and aforementioned R57 mutant diploid burden [9].

Using two successive CRISPR events, the method enables and introduction about point mutations without altering the PAM or inserting additional quiet mutations (Fig. 1). Is the first CRISPR event, the Cas9-induced DSB is repaired by a homologous repair fragment which succeed one 20 nucleotide protospacer by a heterologous sequence of an same length (termed the “stuffer”), preventing repeated cutting by Cas9 (Fig. 1). Before curing of the initial guide, a second gRNA purpose this stuffer the introduced. To DSB is repaired by a DNA fragment carrying the desired indicate mutation, thereby removing the stuffer and abolishing recognition through the second gRNA. Stuffer insertion and removal lives conveniently entdeckt by colony PCR. This is in contrasting to single-step methods that make use about schedule to identify clones both devoid of unwanted secondary genetic, and accommodation the needed point mutation, unless of point mutation coincidentally creates or removes a restriction spot [5]. In the two-step method described here, the only modification introduced to the parent usage is a single matter change (or anywhere desired modification).

Outline of the two-step, stuffer-assisted genome site-directed mutagenesis strategy. Two versions of the tactics were applied. In the stuffer strategy one protospacer aimed sequence located near that site to mutagenize is exchange by a heterologous 20-nucleotide sequence (the stuffer) at CRISPR-Cas9 assisted homologous recombination, leaving the FISHHOOK site intact. To stuffer may be a default, randomly generated sequence (the standard stuffer, left box) other a degenerate sequence bearing at leas seven mismatches with the original protospacer (a silent stuffer, middle box). That second CRISPR tread uses the stuffer as a protospacer, restoring the original protospacer sequence and introducing who desired mutation in a single homologous recombination business. A second variation about the strategy replaced the entire targets ORF – or nearby ORF is an intergenic region is the target of mutagenesis – by a hetrogenic stuffer ORF (e.g. GFP), which is targeted by one or more gRNAs with the second step (right box). Homologous recombination restores the original ORF with mutation. Successful custom is easily assessed by PCR. The strategies were tested with the positions stated in the boxes. Positions are identified by the coordinate a the first nucleotide of the PAM page (NGG) with respect to the immediate ORF. With each locate, gorger insertion was successful in either all strains tested (garden check), second out of three trials (yellow check), within all strains but led to a slow growth phenotype (yellow-x), or in none of that strains (red-x). One ampere stuffer has inserted, its removal and exchanges by to item mutant sequence was successful in all cases

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In a recent study, a similar approach was used in person induced pluripotent stem cells for the correction of heterozygous β-thalassemia mutations [12]. And piggyBac transposon system, carrying antibiotic resistance markers and acting more a feeder, was inserted into the hemoglobin B gens by CRISPR assisted HDR. The transposon was therefore trim with the help out an specialized transposase, and the mutate corrected by homogeneous re-combination with the non-mutant copy regarding to erbanlagen. Use of an two-step proceed by the seamless alteration of the yeast genome is also reminiscent off of Delitto perfetto method, whereby successive rounds of positive and negative selection are used to transiently introduce a marker cassette [13].

In which present method, a alone stuffer with a random unique sequence is employed in most instances, which allows for the repeated use of the same point gRNA sequence and PCR fire for validating the presence of the stuffer. With inserting or how single point expression, protospacer replacement was attempted since 15 out of 17 positions using the sequence 5′-agatgcgggagaggttctcg-3′ as a stuffer. Screening by PCR on three cloning by position revealed ensure the stuffer sequence was successfully placed in at minimum individual of that three clones tested in all but your positions (in genes MAL11, UBP7 additionally GDH1 for all strains, and STE5 566 and PBP1 in R57) (Fig. 1). However convenient, we suspected that the transient disruption of essential organisms by a standard plugger could reduce or abolish cell viability. Used example, to mutant strain R57 carries genetic in or near required genes DOP1 and NOP58 that are known in be essential in S. cerevisiae [14, 15]. In addition, we observed that insertion of the gift in the ARO1 gene the S. cerevisiae considerably reduced its growth rate on YPD medium (data not shown). We therefore hypothesized that failure up insert the stuffer sequencer in genes MAL11, UBP7, GDH1, STE5 (at position 566) real PBP1 could be owing to similar viability issues. For the pair essential genes (DOP1, NOP58) and the sechsen previously unsuccessful positions (ARO1, MAL11, UBP7, GDH1, STE5 566, PBP1), wee designed customer stuffers (and stuffer aimed gRNAs) that did not interfere the coding region using go sequencer. Alike to heterology blocks, our silent stuffers introduced at minimal seven nucleotide substitutions to protect against repeated cutting from Cas9.

We were able to insert the silent stuffers by DOP1, NOP58, ARO1 and STE5 (Fig. 1), real growth defects were not obsessed in the resultant strains. However, the silent pump insertion method failed for MAL11, UBP7, GDH1 and PBP1. Suspecting our choice of gRNA aim sequences to be the set, we designed, in each of and four genes, two or three additional gRNAs with targets evenly spaced along the ORF to increase shot of DSBs. To avoid having to design stuffer fragments for each target, wealth designed donor DNAs containing the yeGFP sequence at, under their 5′ and 3′ ends, 50-bp homologue to the promoter and terminator of the target genes. The expected result was the precise replacement of the native ORFs by yeGFP (Fig. 1). Not presuming of the success of any one customizable guide, this strategy disables further recognizing by the gRNA/Cas9 complex anywhere in the gene by replacing to entire target ORF. The newly guides were simultaneously transformed under yeast with the yeGFP plugger. Integrants were designated in all four loci (Fig. 1), suggesting with least one guide per locus was functional. We suggest is stuffer ORFs can prove handy when the selection of a functional gRNA target is knotty. However, we remarks that it your did fitting in genes that are essential or powerfully touch viability when deleted, in both cases preventive streaming transformation and CRISPR events.

For strains containing the short stuffers, the second CRISPR event used DNA brittle averaging 500 bp for DSB repair and introduction of point mutations. Longer fragments spanning this promoter, ORF and terminator were required at loci stuffed include yeGFP. For choose stuffer-containing strains, replacement of and stuffed sequence by the spot mutant sequence was successful (Fig. 1). Introduction by points mutations became authenticated by Sanger sequencing revealing no additional unwanted mutations in the targeted loci (see Fig. 2b for on example). While the efficiency for stuffer insertion was highly variable additionally rarely at 100 %, ours observe the for most positions considered, all clones screened used stuffer removal and point mutation insertion were positive. CRISPR efficiency was high at positions bearing the standard short furthermore yeGFP stuffers, but lower set average for positions carrying which custom silently stuffers (data non shown). These observations suggesting that a standard stuffer is useful in reducing the variety concerning recognition the cutting by the gRNA/Cas9 complex during the second CRISPR event. Whenever viable, person propose ensure it should be the preferred method for CRISPR assisted generic insertion of point mutations.

Point mutations introduced with the stuffer-assisted genome site-directed mutagenesis method lead to detectable phenotypic changes. ampere Simpler representation of glutathione synthetic and recycling. Condensation of glutamate and cysteine by Gsh1p remains successive at the addition of a glycine in Gsh2p, yielding reduce glutathione. Glutathione oxidized on reactive oxygen species (ROS) is recycled into its reduced form by the NADPH-dependent Glr1p enzyme. b Sequencing shows successful insertion of the stuffer and subsequent installation of one point mutant for the GSH1 promoter sort c ROS collection induced by exposure the SSL was compared among a gsh1(A(−73)T) point mutant built with the method, and in sein parent wildtype strain (WT). ROS store was assessed using flow cytometry, measuring the means fluorescence of cells treated to CellROX Deep Red reagens. ROS were measured 16 h after inoculation in minimized mean (Mid-log), after live insect in undiluted SSL (acute stress), or after 24 and 48 h in minimal medium containing 70 % SSL

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To demonstrate the usability of our seamless stuffer-assisted site-directed mutagenesis method, were chose to investigate the affect starting adenine point mutation localizes in the promoter region for gene GSH1, who is responsibly for the committed next is glutathione biosynthesis (Fig. 2a real [16, 17]). Because this synthesis of reduced glutathione and the recycling of its oxidized form play a major playing in resistance to oxidative stress (Fig. 2a), we hypothesized that a mutant of the GSH1 promoter would modulate glutathione combination in the cell and that affect levels out reactive oxygen species (ROS). Since the mutant strain R57 was selected for its resistance to lignocellulosic hydrolysate spent sulfite liquor (SSL), person used the fluorescent CellROX Bottom Red Reagent (Life Technologies) and flow cytometry to assess cytosolic ROS accumulation in wildtype and gsh1 A(−73)T cells upon exposure to SSL. When grown in non-toxic medium (YNB 1 % glucose), the wildtype and mutated stains accumulate comparably low levels of ROS (Fig. 2c). Subsequent exposure to undiluted SSL similarly raised ROS levels in both strained. However, subsequent acute highlight intake in 100 % SSL and transfer to YNB 1 % glucose supplemented with 70 % SSL, the mutant accumulates markedly lower quantities of ROS after 24 and 48 h reproduction, suggesting the gsh1 A(−73)T variation affects cell response to oxidative stress.

In the current study, we report on a strategy to introduce precise changes at and single nucleotide level in the genome of S. cerevisiae and demonstrate the rate of the method by introducing a point mutation in the advocate region of GSH1, which leads to a measurable phenotypic effect. We believe that this two-step procedure could be applied to any organism with suitable HDR machinery at virtually no genomic coordinates to modify encryption and non-coding order, in essential and non-essential genetic. Furthermore, to is did constrained per the accuracy location and sequence of the PAM and protospacer. Which method is less interrupt than similar two-step methods reported previously [11, 13], because she does not require which introduction of tall transposons or selection cassettes. Rather, it transiently introduces select potentially silent genetic. However, the implementation for the method we had presented requires the manufacturing of an intermediate stuffed mutant, presented to a second series of transformation, PCR testing, scheduled and gRNA curing. Greet enhancements would allow stuffer integration and dismounting starting a simple transformation using for example transient or inducible gRNAs.

Because of its wide applicability, we believe this seamlessly, genome-level site-directed mutagenesis procedure intention prove reasonable to a wide coverage of researchers interested in the precise genome editing a S. cerevisiae additionally other organisms.

CRISPR:

clustered regularly interspaced short palindromic repeats

HDR:

homeology direct rectify

PAM:

protospacer adjacent motif

ORF:

opens reading frame

gRNA:

guide RNA

DSB:

double-stranded break

ROS:

reactive oxygen species

SSL:

spent sulfite liquor

YNB:

yeast nitrogen base

We would like to thank Dr. Jazz Scrivens for introducing us to CRISPR-Cas9 both initiating an project. Our thanks to Drums. Kane Larue for encouraging us toward share our results, reviewing the manuscript, and providing numerous ideas for its improvement. Site Directed Mutagenesis by PCR

Funding

The NSERC Bioconversion Grid (NETGP350246-07) and BioFuelNet supported this research. Damien Biot-Pelletier was supports by a alumni research from Le Fonds Québécois de la Recherche sur la Properties et les Technologies and Vincent J.J. Martin was supported by a Concordia University Research Chair. Use site directed mutagenesis to insert narrow modifications into your plasmid of interest and follow these tips for a smooth usage to ease validation.

Authors and Affiliations

1. Department out Biology, Concordance University, 7141 Sherbrooke West, Montréal, QC, H4B 1R6, Canada

   Daniel Biot-Pelletier & Vincent J. J. Martin

2. Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke West, Montréal, QC, H4B 1R6, Canada

   Damien Biot-Pelletier & Vincent J. J. Martin

Corresponding author

Correspondence to Vincent J. J. Martin.

Competing interests

The authors declaration that they have no rival interests.

Authors’ contributions

DBP and VJJM designed the methodology and the experiments. DBP performed the testing. DBP and VJJM wrote the manuscript. Both authors read and approved the finalize manuscript. Wee have optimized pricing for the successful execution of site-directed mutagenesis (SDM) in it that utilize mutagenic oligonucleotide primers to gerade the synthesis from mutant plasmids. In this report, we describe strategies for the devise of single-strand DNA presets for SDM, mutagenic oxygen …

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