CRISPR-Cas9: a Powerful Tool to Efficiently Engineer Saccharomyces

life

Review CRISPR-Cas9: A Powerful Tool to Efﬁciently Engineer Saccharomyces cerevisiae

João Rainha , Joana L. Rodrigues and Lígia R. Rodrigues *

Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal; [email protected] (J.R.); [email protected] (J.L.R.) * Correspondence: [email protected]; Tel.: +351-253601978

Abstract: Saccharomyces cerevisiae has been for a long time a common model for fundamental biological studies and a popular biotechnological engineering platform to produce chemicals, fuels, and pharmaceuticals due to its peculiar characteristics. Both lines of research require an effective editing of the native genetic elements or the inclusion of heterologous pathways into the yeast genome. Although S. cerevisiae is a well-known host with several molecular biology tools available, a more precise tool is still needed. The clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9) system is a current, widespread genome editing tool. The implementation of a reprogrammable, precise, and speciﬁc method, such as CRISPR-Cas9, to edit the S. cerevisiae genome has revolutionized laboratory practices. Herein, we describe and discuss some applications of the CRISPR-Cas9 system in S. cerevisiae from simple gene knockouts to more complex processes such as artiﬁcial heterologous pathway integration, transcriptional regulation, or tolerance engineering.

Keywords: CRISPR-Cas9; CRISPR-Cas9 applications; genome editing; Saccharomyces cerevisiae

1. Introduction Saccharomyces cerevisiae has long been a common model for fundamental biological studies because of its safety and ease of handling. This yeast has been extensively used to Citation: Rainha, J.; Rodrigues, J.L.; elucidate eukaryotic processes from basic metabolism to protein/gene interaction or even Rodrigues, L.R. CRISPR-Cas9: A Powerful Tool to Efficiently Engineer evolution [1]. Moreover, it is also a popular biotechnological engineering platform used Saccharomyces cerevisiae. Life 2021, 11, to produce chemicals, fuels, and pharmaceuticals [2]. Associated with these two lines of 13. https://dx.doi.org/10.3390/ research is the need to effectively edit the native genetic elements or introduce heterologous life11010013 pathways into the yeast genome. For instance, for metabolic engineering purposes, several genetic modifications are required besides the introduction of heterologous genes in order to Received: 16 November 2020 stream the metabolic fluxes toward the production of the desired product. The development Accepted: 24 December 2020 of an efficient cell factory is associated with several native gene deletions, overexpression, Published: 26 December 2020 or replacements. All these modifications require a cycle of individual transformation, selection, and confirmation, thus making the process very time-consuming. Furthermore, Publisher’s Note: MDPI stays neu- each cycle is associated with the integration of a selective marker. However, the number of tral with regard to jurisdictional claims markers available is limited, which also limits the number of sequential modifications that in published maps and institutional can be performed. affiliations. S. cerevisiae is known to possess a very efficient homologous recombination (HR) machinery [3]. Researchers have been taking advantage of this feature for in vivo assembly of multiple linear fragments instead of using in vitro molecular biology techniques. Most of

Copyright: © 2020 by the authors. Li- the in vivo assembly performed in yeast was focused on circular vector construction. censee MDPI, Basel, Switzerland. This However, for metabolic engineering purposes, genomic integration offers a more stable article is an open access article distributed expression system and eliminates the need of ensuring a selective pressure for plasmid under the terms and conditions of the maintenance. Nevertheless, in vivo assembly in combination with genomic integration is Creative Commons Attribution (CC BY) associated with very low efﬁciencies [4]. license (https://creativecommons.org/ Double-strand breaks (DSBs) in the genome occur due to several environmental factors licenses/by/4.0/). and can be repaired by HR or nonhomologous end-joining (NHEJ). In S. cerevisiae it is

Life 2021, 11, 13. https://dx.doi.org/10.3390/life11010013 https://www.mdpi.com/journal/life Life 2020, 10, x FOR PEER REVIEW 2 of 17

maintenance. Nevertheless, in vivo assembly in combination with genomic integration is Life 2021, 11, 13 associated with very low efficiencies [4]. 2 of 16 Double-strand breaks (DSBs) in the genome occur due to several environmental factors and can be repaired by HR or nonhomologous end-joining (NHEJ). In S. cerevisiae it is known that the dominant repair is performed by HR. The introduction of a DSB has been shown to knownincrease that the theefficiency dominant of homologous repair is performed integration by HR.of linear The introductionDNA fragments of a DSB has been shown to increase the efficiency of homologous integration of linear DNA fragments with with homologous ends [5]. These features led researchers to develop methods that use homologous ends [5]. These features led researchers to develop methods that use DSBs for DSBs for site-directed genome editing. Theoretically, these methods could be used for site-directed genome editing. Theoretically, these methods could be used for marker-free marker-free modifications and examples include zinc finger nucleases (ZFN) and tran- modifications and examples include zinc finger nucleases (ZFN) and transcription-activator- scription-activator-like effector nucleases (TALEN). Both methods involve the design of like effector nucleases (TALEN). Both methods involve the design of proteins that recognize proteins that recognize a specific DNA sequence and cause a site-specific DSB through a specific DNA sequence and cause a site-specific DSB through DNA–protein interaction. DNA–protein interaction. The DSB is then repaired by HR with a provided DNA fragment The DSB is then repaired by HR with a provided DNA fragment with homologous ends. with homologous ends. However, a new ZFN or TALEN protein has to be engineered for However, a new ZFN or TALEN protein has to be engineered for each target modification, each target modification, making these methods also very laborious [6,7]. making these methods also very laborious [6,7]. Evolution allowed bacteria to develop several systems to degrade foreign DNA as a Evolution allowed bacteria to develop several systems to degrade foreign DNA as a de- defense mechanism. The most well-known are restriction enzymes, which rapidly became fense mechanism. The most well-known are restriction enzymes, which rapidly became the the “workhorses“workhorses of molecular of molecular biology” biology”[8]. More [ 8recently,]. More recently,another anotherprokaryotic prokaryotic immune immune system system was identifiedwas identified [9]. This [9 ].system This systemconsists consists of clustered, of clustered, regularly regularly interspaced, interspaced, short short palin- palindromic repeatsdromic (CRISPR) repeats (CRISPR) and CRISPR and-assoc CRISPR-associatediated nucleases nucleases (CRISPR-Cas), (CRISPR-Cas), widely widely dis- distributed amongtributed bacteria among and bacteriaarchaea and[10].archaea CRISPR [-10Cas]. has CRISPR-Cas the function has of the recognizing function of recognizing and degradingand invading degrading nucleic invading acids. The nucleic system acids. is divided The system in three is divided classes in (I, three II, and classes III) (I, II, and III) based on the casbased gene on sequences, the cas gene operon sequences, organization operon, and organization, the repeats and within the repeats CRISPR within CRISPR arrays [11]. Thearrays increased [11]. Theknowledge increased of knowledgeCRISPR action of CRISPR mechanism action broke mechanism new ground broke new ground regarding a possibleregarding applicability a possible for applicability this system for to thisedit systemgenetic toelement edit genetics of an elements organism. of an organism. Due to their simplicityDue to theirrelative simplicity to other relativeclasses, toclass other II CRISPR classes,-Cas class systems II CRISPR-Cas have the systems have most well-developedthe most methods well-developed for genomic methods engineering for genomic nowadays. engineering In class II, nowadays. all the func- In class II, all the tions of the effectorfunctions complex of the are effector performed complex by a aresingle performed protein by[12] a. singleThe following protein [com-12]. The following ponents are required:components the RNA are required:-guided nucl the RNA-guidedease Cas9, a CRISPR nuclease RNA Cas9, (crRNA), a CRISPR an RNA auxil- (crRNA), an aux- iary trans-activatingiliary trans-activatingcrRNA (tracrRNA), crRNA and (tracrRNA),an RNase III. andThe anaction RNase mechanism III. The actionis sim- mechanism is ple and aims atsimple the creation and aims of ata DSB the creationin the target of a DSB DNA. in theA hybrid target DNA.of the ARNA hybrid molecules of the RNA molecules directs Cas9 todirects the target Cas9 DNA to the site target and DNAthe Cas9 site cleaves and the a Cas9targeting cleaves DNA a targeting sequence DNA con- sequence containing a protospacertaining-adjacent a protospacer-adjacent motif (PAM) sequence. motif (PAM) Jinek sequence. et al. [13] Jinek postulated et al. [ 13that] postulated the that the requirement forrequirement RNase and fortracRNA RNase can and be tracRNA bypassed can by be fusion bypassed crRNA by fusionand tracrRNA crRNA andto tracrRNA to form a single-guideform aRNA single-guide (gRNA) RNA simplifying (gRNA) the simplifying process and the processapplication. and application. The gRNAs The gRNAs are are composed composedof a homologous of a homologous sequence sequence 20 nt upstream 20 nt upstream of the ofPAM the PAMsequence sequence (guide (guide sequence) sequence) and andthe scaffolding the scaffolding loop loop structure structure to attach to attach the Cas9 the Cas9 (Figure (Figure 1). 1).

Figure 1. The mechanism of action of clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR- Cas9). The guide RNA (gRNA) molecule directs Cas9 protein to the target DNA and Cas9 cleaves genomic DNA 3–4 bp Figure 1. The mechanism of action of clustered, regularly interspaced, short palindromic upstream of the PAM (protospacer-adjacent motif) site. repeats–associated Cas9 (CRISPR-Cas9). The guide RNA (gRNA) molecule directs Cas9 protein to the targetThe DNA type and II CRISPR-Cas9Cas9 cleaves genomic system was DNA implemented 3–4 bp upstream in S. cerevisiae of the PAMfor genetic modiﬁca- (protospacer-adjacenttion purposes motif) bysite. DiCarlo et al. [14]. The researchers demonstrated that co-transforming a gRNA targeting a negative selectable marker together with a 90-bp double-stranded HR donor with a frameshift mutation in the targeted reading frame and PAM replacement by a stop codon resulted in almost 100% of mutated cells. Life 2021, 11, 13 3 of 16

In this review, we discuss in detail the CRISPR-Cas9 system components with a focus on the S. cerevisiae chassis, as well as the recent CRISPR-Cas9-based applications.

2. The CRISPR-Cas9 System Components 2.1. Cas9 Protein The Cas9 protein from Streptococcus pyogenes (SpCas9) fused to a nuclear tag is the most used Cas9 protein in S. cerevisiae. The chosen Cas9 influences the guide RNA design because Cas9 from different origins recognize different PAM sequences. For instance, SpCas9 recognizes the PAM sequence (5’–3’) NGG. The DNA sequence of Cas9 used in yeast can be native, codon-optimized for yeast or even for humans [14]. Regarding the expression system, usually Cas9 is expressed under the control of constitutive promoters from self- replicating centromeric (e.g., [14]) or 2µ (e.g., [15]) plasmids. Other studies integrated the Cas9 in yeast genome (e.g., [16]). Cas9 toxicity has been reported in some experiments, however this can be easily bypassed using weaker or inducible promoters [14,17]. The Cas9 expression system can be chosen to fit the particularities of the research. For instance, a plasmid-based strategy is preferred for single modifications because the plasmid can be easily cured after the process. On the other hand, for multiple modifications, the genomic integration offers a more stable platform for Cas9 expression.

2.2. Guide RNA The key step for the development of an efficient CRISPR-Cas9 system to target a desired genomic sequence is the design, expression, and delivery of the RNA components. As already mentioned, in S. cerevisiae the most popular strategy has been the expression of a single chimeric RNA molecule named gRNA. The gRNA combines the functions of crRNA and a tracRNA in a single RNA complex making the construction and application easier. To ensure the right function of gRNA to form a Cas9/gRNA complex, both ends must be accurately defined. For the expression of gRNA in S. cerevisiae, RNA polymerase III promoters are commonly used. The expression cassette containing the small nucleolar RNA (SNR52) promoter, an RNA polymerase III promoter, and the SUPpressor (SUP4) terminator was used by DiCarlo et al. [14] to functionally express the gRNA in a laboratory yeast strain with engineering efficiencies reaching 100%. The same system was also used in industrial strains with efficiencies ranging between 65% and 78% [18]. Nevertheless, other cassette setups were also used yielding good results. For instance, Ryan et al. [19] expressed the gRNA fused to a Hepatitis delta virus ribozyme controlled by a tRNA promoter and SNR52 terminator. The efficiency was almost 100% in a laboratory strain and 90% in an industrial strain. It was observed that the presence of the fused ribozyme increased the number of synthesized gRNAs. The authors reasoned that the ribozyme would protect the 5’ end of gRNA from endogenous 5’ exonucleases resulting in higher efficiencies. RNA polymerase II promoters were also used achieving 100% efficiency [20]. In contrast, other researchers separated the expression of the targeting crRNA and tracRNA instead of using the chimeric gRNA. Both RNA molecules were transcribed by different RNA polymerase III promoters. The efficiencies obtained ranged from 75% to 100% [15]. The gRNA molecule is composed by a scaffolding sequence, necessary for Cas9 binding, and by the guide sequence. The design of a gRNA to target a defined locus requires the ~20 bp guide sequence upstream of the selected PAM sequence to direct Cas9. The vector construction containing the desired gRNA is another challenge in the development of CRISPR systems. Most studies use PCR to insert the gRNA sequence using phosphorylated primers containing the target-specific 20 bp sequence to amplify the whole vector followed by recircularization via ligase [21]. Cloning methods such as circular polymerase extension cloning (CPEC), which uses polymerase to join overlapping DNA fragments can also be used to clone gRNA in a plasmid [22]. Fragment assembly is another possible approach, these can be accomplished by in vivo assembly [17] or by in vitro methods such as the Gibson assembly [23]. In addition, restriction enzymes located between the promoter and the gRNA scaffolding sequence can also be used [24]. Life 2020, 10, x FOR PEER REVIEW 4 of 17

extension cloning (CPEC), which uses polymerase to join overlapping DNA fragments Life 2021, 11, 13 can also be used to clone gRNA in a plasmid [22]. Fragment assembly is another possible4 of 16 approach, these can be accomplished by in vivo assembly [17] or by in vitro methods such as the Gibson assembly [23]. In addition, restriction enzymes located between the pro- Tomoter avoid and PCR the ampliﬁcations, gRNA scaffolding the sequence gRNA cassette can also can be be used synthesized [24]. To avoid and PCR integrated amplifica- in vectorstions, the using gRNA for instance cassette Uracil-Speciﬁccan be synthesized Excision and Reagentintegrated (USER) in vectors cloning using [25] for or Goldeninstance GateUracil Assembly-Specific [Excision15] (Figure Reagent2). (USER) cloning [25] or Golden Gate Assembly [15] (Fig- ure 2).

Figure 2. Molecular biology techniques for the construction of guide RNA (gRNA) carrying plas- Figure 2. Molecular biology techniques for the construction of guide RNA (gRNA) carrying plasmids.mids. gRNA gRNA plasmids plasmids can can be be constructed constructed using using traditional traditional methods methods such such as digestion–ligationas digestion–ligation using us- restrictioning restriction enzymes enzymes and T4 and ligase T4 ligase (A) or ( byA) PCRor by based PCR methodsbased methods such as such circular as circular polymerase polymerase exten- sionextension cloning cloning (CPEC) (CPEC) (B). Homology-recombination-based (B). Homology-recombination methods-based methods are other are possible other approaches. possible ap- Homologyproaches. recombinationHomology recombination can be performed can be by performedin vivo assembly by in vivo using assemblyS. cerevisiae usingmachineryS. cerevisiae (C)ma- or usingchinery in vitro(C) o methodsr using in such vitro as methods Gibson assemblysuch as Gibson (D). assembly (D).

SinceSince any any ~20 ~20 bpbp sequence sequence nextnext toto aa PAM PAM site site can can be be used used in in the the gRNA gRNA design, design, the the biggestbiggest concernconcern isis toto ensure that the the Cas9 Cas9 will will not not recognize recognize an an unwanted unwanted sequence sequence in inthe thegenome genome leading leading to off to off-target-target effects. effects. The The off off-target-target effect, effect, when when a DNA a DNA site site other other than than the thetarget target is cleaved is cleaved by by Cas9, Cas9, must must be be considered considered when when performing performing genetic genetic modifications modifications us- usinging the the CRISPR CRISPR-Cas9-Cas9 system. system. Other Other factors factors may may influence influence the the gRNA gRNA design design and and may may vary varyaccording according to the to desired the desired application. application. For instance, For instance, for gene for gene knockout, knockout, the beginning the beginning of an ofopen an open reading reading frame frame (ORF) (ORF) is preferred is preferred to insert to insert a STOP a STOP codon codon to precociously to precociously stop stoptran- transcriptionscription avoiding avoiding any any protein protein translation. translation. Regarding Regarding the choice the choice of guide of guide sequence, sequence, there thereis a wide is a wide collection collection of online of online tools toolsavailable available to help to researchers help researchers design design a gRNA a gRNA for a target for a targetgenomic genomic sequence. sequence. The tools The toolsaim to aim minimize to minimize the offthe-target off-target effects effects by bymatching matching the the de- desiredsired sequence sequence against against the the reference reference genome genome assigning assigning a ascore score based based on on the the specificity specificity of ofthe the guide guide sequence sequence or orby by looking looking for for potential potential PAM PAM sites sites with with minimized minimized possible possible off- off-targets in a given DNA sequence. Some of the tools also provide other potential re- quirements to ensure an efficient DSB, such as the GC content and self-complementary or poly T presence in the gRNA molecule, calculating a score also based on these features. Life 2021, 11, 13 5 of 16

Some available webtools include, CRISPy (www.crispy.secondarymetabolites.org)[26], CRISPR-ERA (www.crispr-era.stanford.edu)[27], E-CRISPR (www.e-crisp.org)[28], Bench- ling (www.benchling.com), or ATUM gRNA design tool (www.atum.bio). However, since S. cerevisiae has a high HR capability and NHEJ rarely occurs, the chances of off-target effects is drastically reduced as confirmed in some works [14,21]. Nevertheless, the high efficiencies reported are only applied for laboratorial yeast strains, which are usually haploid or diploid homozygotic making possible the performance of markerless genetic modifications carried out by the CRISPR-Cas9 system. In heterozygous organisms, such as polyploid industrial S. cerevisiae strains or other eukaryotic organisms, a gRNA can be designed for an allele-specific target if a loci has different PAM sites [29]. How- ever, the presence of an intact chromosome could facilitate the HR repair competing with the editing event in a phenomenon called loss of heterogenicity (LOH). De Vries et al. [30] observed that the editing efficiency can decrease by two orders of magnitude when targeting only one of two homologous chromosomes in a heterozygous S. cerevisiae. The authors suggested that gRNAs should target homozygous stretches in heterozygous genomes, and markers should be used when allele-specific gene editing is required. Methods for the identification of CRISPR-Cas9 off-target sites have been developed and are reviewed elsewhere [31]. However, these types of studies are usually employed in relatively more complex genomes such as mammalian cells. Notwithstanding, Waldrip et al. [32] used cross-linking chromatin immunoprecipitation (ChIP) sequencing to detect off-target sites in S. cerevisiae and found that, as expected, Cas9 is highly specific and virtually lacks off-target sites within the yeast genome.

2.3. Donor DNA As mentioned, the dominant mode of DSB repair in yeast is HR. In order to efficiently apply a CRISPR-Cas9 system to a desired genomic target, it is necessary to supply the homologous donor template carrying the desired modification. The use of short single- strand [17] or double-strand [14] DNA donor oligos can act as the simplest repair template. The lengths on donor DNA vary depending on the type of desired modification, from a STOP codon insertion to a complete heterologous biosynthetic pathway. It is very important to ensure that, after recombination, the PAM site is removed from the target to prevent continuous cutting by Cas9. Regarding the donor delivery, it can be as part of an expression vector [15] or as simple linear DNA oligos. The CRISPR system was also combined with in vivo assembly of various DNA fragments eliminating the need of cloning processes. For instance, a metabolic pathway of six genes and 300 bp homology arms consisting in four DNA fragments was assembled in vivo and used as a DSB repair template by Tsai et al. [33]. Three DNA oligos carrying a genetic pathway and 1 kb homologous arms were used by Apel et al. [23] resulting in 40% efficiency in a determined locus. Although versatile and faster, the combination of donor in vivo assembly with CRISPR-Cas9 DSB repair is associated with low efficiencies. Pre-assemble of the DNA fragments always leads to higher efficiencies [34]. Plasmids containing combined gRNA and a DNA repair template can also be used [35]. Given that during gRNA or crRNA maturation, the 5’ end region is cleaved by 5’ exonuclease and other endogenous nucleases, the researchers hypothesized the use of this region to harbor the HR disruption donor DNA, as seen in some works [15]. This is advantageous when the combined DNA elements are small enough to be synthesized and inserted into the CRISPR plasmid. Regarding the length of homology arms, the minimal homology required is not directly related with the CRISPR-Cas9. Early studies in genetic integration defined 30 bp minimal homology at each end of a DNA fragment for recombination [36]. However, higher homology arms (200 to 1000 bp) result in high yield of recombination efficiencies mainly when large heterologous pathways need to be integrated. Life 2021, 11, 13 6 of 16 Life 2020, 10, x FOR PEER REVIEW 6 of 17

3. CRISPR-Cas9 Applications in Saccharomyces cerevisiae Knockin and Knock3.out CRISPR-Cas9 Applications in Saccharomyces cerevisiae Simple knockin3.1. Knockin or knockout and Knockout of genes are routinely used in S. cerevisiae studies of fundamental metabolismSimple or also knockin to develop or knockoutnovel strains of genes adapted are to routinely produce used useful in chemi-S. cerevisiae studies of cals. These modificationsfundamental are easily metabolism achieved or by also conventional to develop gene novel recombination, strains adapted due toto produce useful the high HR capabilitychemicals. of this These yeast modifications [37], however are they easily are achievedinherent to by the conventional use of selective gene recombination, markers. Althoughdue recombinase to the high HR-based capability methods of such this yeastas Cre/LoxP [37], however or Flp/FRT they ha areve inherent been to the use of extensively usedselective in marker markers. recycling Although [38,39], recombinase-basedthey are time-consuming methods since such they as require Cre/LoxP or Flp/FRT another transformationhave been cycle extensively to remove used the in marker. marker In recycling addition, [38 these,39], theymethods are time-consuming have the since they drawback of leavrequireing a copy another of a transformationrepeat sequence cycle in the to genome. remove theThese marker. “scars” In may addition, lead these methods to genome instabilityhave theafter drawback multiple of rounds leaving of a marker copy of arecovery repeat sequence [40]. The in development the genome. Theseof “scars” may the CRISPR-Cas9lead system to genome allowed instability the researchers after multiple to perform rounds high of- markerefficiency recovery knockin [40 and]. The development knockout withoutof the CRISPR-Cas9use of selection system markers. allowed Different the researchers studies hav toe perform provedhigh-efficiency that single knockin and and multiple geneknockout disruptions/integration without the use ofs can selection be achieved markers. by Differentthe combinatorial studies have effects proved that single of high-efficiencyand HR multiple and CRISPR gene disruptions/integrationssystem in a markerless way. can beThe achieved system byhas the proven combinatorial to effects of be very efficienthigh-efficiency with values ranging HR and up CRISPR to 100%. system in a markerless way. The system has proven to be Regarding verythe knockout efficient with of genes, values Generosoranging up et to al. 100%. [17], for instance, designed a CRISPR-Cas9 systemRegarding to create a the DSB knockout in the threonine of genes, Generosodeaminase et al.(ILV) [17 ],gene. for instance, The single designed- a CRISPR- stranded donor Cas9DNA systemused for to recombination create a DSB in was the composed threonine deaminaseof 40 nt upstream (ILV) gene. and 4 The0 nt single-stranded downstream of thedonor ILV DNA ORF, used the knockout for recombination was 100% was efficient. composed No marker of 40 nt upstreamintegration and was 40 nt downstream of the ILV ORF, the knockout was 100% efficient. No marker integration was required to required to delete an entire ORF. For gene knockout, besides deleting an entire ORF, other delete an entire ORF. For gene knockout, besides deleting an entire ORF, other strategies strategies can be used such as frameshift modification or insertion of a STOP codon (Fig- can be used such as frameshift modification or insertion of a STOP codon (Figure3). ure 3). To knockout the canavanine resistance (CAN1) gene, Bao et al. [15] caused a 8 bp To knockout the canavanine resistance (CAN1) gene, Bao et al. [15] caused a 8 bp deletion deletion leading to a frameshift resulting in CAN1 knockout by putting a STOP codon in leading to a frameshift resulting in CAN1 knockout by putting a STOP codon in the frame the frame in the beginning of the gene. The 8 bp included the PAM site and the last 3 bp in the beginning of the gene. The 8 bp included the PAM site and the last 3 bp of the gRNA of the gRNA to prevent the continuous recognition and cleavage by the CRISPR-Cas after to prevent the continuous recognition and cleavage by the CRISPR-Cas after recombination. recombination.

Figure 3. Strategies used to gene knockout using double-strand breaks (DSB) originated by clustered, regularly interspaced, Figure 3. Strategies used to gene knockout using double-strand breaks (DSB) originated by clus- short palindromictered, regularly repeats–associated interspaced, Cas9short (CRISPR-Cas9).palindromic repeats The– knockoutassociated can Cas9 be performed(CRISPR-Cas9). by providing The knock- a homologous repair fragmentout can for be the performed deletion by of anproviding entire gene a homologous (A) or to cause repair a frameshiftfragment for modification the deletion (B of). an entire gene (A) or to cause a frameshift modification (B). Multiple gene disruptions can be used for gene characterization in complex genetic Multiple genepathways disruptions [41] and can also be used to facilitate for gene the characterization development of in an complex efficient chassisgenetic for heterologous pathways [41] andproduction also to facilitate [42]. Sequential the development gene disruptions of an efficient are traditionally chassis for achieved heterolo- by the replacement gous productionof [42] the. targetSequential gene withgene a disruptions selectable marker are traditionally through HR achieved in a sequential by the manner. re- The markers are usually recycled by different systems, which is a very laborious process. CRISPR-Cas9 placement of the target gene with a selectable marker through HR in a sequential manner. can bypass this limitation, however there are some problems associated with multiple gR- The markers are usually recycled by different systems, which is a very laborious process. NAs’ expression, mainly the need to use various RNA polymerase III regulatory elements CRISPR-Cas9 can bypass this limitation, however there are some problems associated to express them. Bao et al. [15] developed a CRISPR system to overcome this limitation. with multiple gRNAs’ expression, mainly the need to use various RNA polymerase III For that purpose, the expression of three gRNAs was carried by a single promoter and regulatory elements to express them. Bao et al. [15] developed a CRISPR system to over- the sequences were separated by direct repeats. In a different experiment, in order to come this limitation. For that purpose, the expression of three gRNAs was carried by a knockout three genes in a single step, Generoso et al. [17] expressed the gRNAs separately, single promoter and the sequences were separated by direct repeats. In a different exper- also obtaining good results. Jakoˇcinaset al. [21] developed a CRISPR system for quintuple iment, in order to knockout three genes in a single step, Generoso et al. [17] expressed the gRNA expression in a single plasmid controlled by a single SNR52 promoter. Good efficien- gRNAs separately, also obtaining good results. Jakočinas et al. [21] developed a CRISPR

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system for quintuple gRNA expression in a single plasmid controlled by a single SNR52 cies were reported, however the transformation efficiency decreased with the number of promoter. Good efficiencies were reported, however the transformation efficiency de- expressed gRNAs. Liu et al. [43] expressed three gRNAs in a single cassette and achieved creased with the number of expressed gRNAs. Liu et al. [43] expressed three gRNAs in a triple disruption efficiencies of 95%. Therefore, for the expression of multiple gRNAs using single cassette and achieved triple disruption efficiencies of 95%. Therefore, for the ex- a single RNA polymerase promoter, each gRNA should be flanked by cleavage sequences pression of multiple gRNAs using a single RNA polymerase promoter, each gRNA should such as direct RNA repeats [15]; self-cleavable ribozyme sequences [19,20] or by RNA pro- be flanked by cleavage sequences such as direct RNA repeats [15]; self-cleavable ribozyme cessing sequences such as tRNA. Zhang et al. [44] developed a multiplex CRISPR system sequences [19,20] or by RNA processing sequences such as tRNA. Zhang et al. [44] devel- expressing gRNA–tRNA arrays with a single RNA polymerase promoter. The researchers reportedoped a multiplex a simultaneous CRISPR disruption system expressin of eight genes,g gRNA which–tRNA is currentlyarrays with the a highest single multiplexRNA pol- editionymerase reported promoter. in yeast.The researchers reported a simultaneous disruption of eight genes, whichDirect is currently point mutationsthe highest (single-nucleotide multiplex edition reported changes) in can yeast. also be performed using CRISPR-Cas9.Direct point Mans mutations et al. [16 (single] efficiently-nucleotide caused changes) point mutations can also inbe specificperformed genes using us- ingCRISPR a CRISPR-Cas9. approach.Mans et al. This [16] kind efficiently of approach caused can point be mutations useful for studiesin specific of thegenes biological using a significanceCRISPR approach. of mutations This kind caused of approach by artificial can evolution, be useful sincefor studies not all of mutations the biological obtained signif- in thisicance kind of ofmutations experiments caused contribute by artificial to a evolution, desired phenotype since not [ 45all]. mutations obtained in this kindAs of experiments mentioned, thecontribute CRISPR-Cas9 to a desired system phenotype may be tremendously[45]. useful to knockout singleAs or mentioned, even multiple the genes CRISPR without-Cas9 thesystem requirement may be oftremendously a selection marker. useful Theto knockout knockin ofsingle genes or oreven other multiple genomic genes components without the is also require an importantment of a elementselection in marker.S. cerevisiae The knockingenetic studies.of genes Theor other knockin genomic of genetic components parts is usefulis also bothan important for the development element in S. of cerevisiae yeast cell genetic facto- riesstudies. and forThe fundamental knockin of genetic studies. parts Therefore, is useful knockin both for events the maydevelopment include the of integrationyeast cell fac- of heterologoustories and for genes,fundamental the replacement studies. Therefore, of genes byknockin a feedback-insensitive events may include orthologue the integration or the promoterof heterologous replacement genes, tothe adjust replacement the expression of genes of by a specific a feedback gene-insensitive (Figure4). orthologue or the promoter replacement to adjust the expression of a specific gene (Figure 4).

Figure 4. Strategies used to integrate DNA fragments using double-strand breaks (DSB) originated by clustered, regularly Figure 4. Strategies used to integrate DNA fragments using double-strand breaks (DSB) originated by clustered, regularly interspaced,interspaced, shortshort palindromicpalindromic repeats–associatedrepeats–associated Cas9Cas9 (CRISPR-Cas9).(CRISPR-Cas9). The knockin can be performedperformed byby providingproviding aa homologoushomologous repairrepair fragmentfragment forfor genegene integrationintegration ((AA),), aa genegene replacementreplacement ((BB),), oror thethe replacementreplacement ofof regulatoryregulatory elementselements suchsuch asas aa promoterpromoter ((CC).).

Combining gene disruption disruption with with the the integration integration of ofa desired a desired component component may may be ad- be advantageousvantageous since since it itmakes makes it itpossible possible to to perform perform various various modifications modiﬁcations in in a a single step.step. For example,example, MansMans et et al. al. [16 [16]] developed developed a CRISPR-Cas9 a CRISPR-Cas9 system system to target to target an undesired an undesired gene andgene simultaneously and simultaneous transformly transform the donor the donor DNA DNA repair repair with otherwith other ﬁve DNA five DNA fragments fragments with with homologous arms to in vivo combine the fragments and replace the undesired ORF.

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Life 2020, 10, x FOR PEER REVIEW homologous arms to in vivo combine the fragments and replace the undesired ORF. This8 of 17 kind of approach (Figure5) is very useful for the development of yeast cell factories as discussedThis kind below.of approach (Figure 5) is very useful for the development of yeast cell factories as discussed below.

. Figure 5. Combination of in vivo recombination and fragment integration using double-strand breaks Figure 5. Combination of in vivo recombination and fragment integration using double-strand (DSB)breaks originated (DSB) originated by clustered, by clustered, regularly regularly short palindromic short palindromic repeats–associated repeats–associated Cas9 (CRISPR-Cas9). Cas9 The(CRISPR homologous-Cas9). repairThe homologous fragment can repair be in fragment vivo assembled can be usingin vivoS. assembled cerevisiae machinery using S. cerevisiae by providing ma- thechinery fragments by providing with homologous the fragments ends. with The homologous assembled fragment ends. The is usedassembled to homology fragment repair is used the DSB.to homology repair the DSB. 3.2. Cell Factory Development 4. CellThe F designactory D andevelo constructionpment of microbial cell factories is more than ever an attractive approach to develop bioengineering processes to produce chemicals, fuels, or pharmaceu- The design and construction of microbial cell factories is more than ever an attractive tics. Furthermore, S. cerevisiae is an appealing model for bioengineering processes because approach to develop bioengineering processes to produce chemicals, fuels, or pharmaceu- allied with its rapid production cycles, it is easy to maintain, to manipulate, and is suitable tics. Furthermore, S. cerevisiae is an appealing model for bioengineering processes because for large-scale fermentation. The molecular “tools” available have helped the researchers to allied with its rapid production cycles, it is easy to maintain, to manipulate, and is suitable make yeast cell factories increasingly adapted to produce a desired product [46]. The devel- for large-scale fermentation. The molecular “tools” available have helped the researchers opment of efficient cell factories is associated with several genetic modifications, since not to make yeast cell factories increasingly adapted to produce a desired product [46]. The only the insertion of the heterologous genes is required but also the rewiring of the carbon development of efficient cell factories is associated with several genetic modifications, flux toward the precursor synthesis or even other convenient modifications to improve the since not only the insertion of the heterologous genes is required but also the rewiring of process. The CRISPR-Cas9 system appeared as a “Swiss army knife” [16] in the researcher’s the carbon flux toward the precursor synthesis or even other convenient modifications to molecular toolbox because it allowed the performance of several genetic modifications in a fastimprove and efficient the process. way. Furthermore,The CRISPR- theCas9 requirement system appeared of a constant as a “ selectiveSwiss army pressure knife” during [16] in fermentationthe researcher’s is negatedmolecular since toolbox CRISPR-Cas9 because it allows allowed a markerlessthe performance genetic of manipulation.several genetic Inmodifications addition, several in a fast design–build–test and efficient way. cycles Furthermore are always, the necessary, requirement which of increasesa constant the se- numberlective pressure of modifications during fermentation required [6,7 ].is Bynegated using since CRISPR-Cas9 CRISPR-Cas9 systems, allows genetic a markerless editing hasgenetic become manipulation. multiplex possible,In addition, which several may design significantly–build decrease–test cycles the are time always to perform necessary the , designedwhich increases genetic the modifications number of formodifications a cell factory required construction. [6,7]. By The using flexibility CRISPR of- CRISPR-Cas9 sys- Cas9tems, allied genetic with editing the researcher’s has become inventiveness multiplex possible, resulted which in the may development significantly of systems decrease that the facilitatetime to perform and accelerate the designed metabolic genetic engineering modifications processes. for Researchers a cell factory are constru not onlyction. focused The onflexibility developing of CRISPR new CRISPR-based-Cas9 allied with strategies the researcher’s for direct inventiveness genome engineering resulted in but the also devel- on applyingopment of CRISPR-Cas9 systems that to facilitate improve and other accelerate conventional metabolic molecular engineering biology processes. lab practices, Re- speedingsearchers up are the not strain only buildingfocused on process. developing For example, new CRISPR Li et- al.based [47 ]strategi developedes for a direct CRISPR ge- systemnome engineering capable of sequential but also on rounds applying of gene CRISPR targeting-Cas9 withto improve simultaneous other conventional gRNA plasmid mo- curinglecular mediated biology lab by practices CRISPR-Cas9., speeding Moreover, up the instrain Zhang building et al.’s process. work [44 For], the example, developed Li et CRISPRal. [47] developed method skips a CRISPR the Escherichia system coli capabletransformation of sequential and rounds verification of gene steps. targeting The flexibil- with itysimultaneous and simplicity gRNA of the plasmid CRISPR-Cas9 curing mediated system made by CRISPR it well accepted-Cas9. Moreover by genetic, in engineers Zhang et foral.’s the work development [44], the developed of yeast cell CRISPR factories method (Table1 ).skips the Escherichia coli transformation and verification steps. The flexibility and simplicity of the CRISPR-Cas9 system made it well accepted by genetic engineers for the development of yeast cell factories (Table 1). For genetic engineering purposes, the use of plasmids brings the advantage of expressing a heterologous protein/pathway in a multi-copy way once a 2µ plasmid is maintained at 10–50 copies per cell. However, genome integration has several benefits over plasmids because it allows gene expression stability and lower heterogeneity and eliminates the requirement of using selective growth media after confirmation. Shi et al. [48]

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Table 1. Some CRISPR-based applications in Saccharomyces cerevisiae. ARS: autonomously replicating sequence; CEN: yeast centromere.

Cas Protein Transformed Number of Objective gRNA Expression Phenotype Desired Ref. Expression System Elements Editions Genome editing CEN plasmid pSNR52 Canavanine resistance CRISPR-Cas9 plasmid 1 [14] pSNR52 plus CRISPR Hydrocortisone CRISPR-Cas9 plasmid Genome editing 2µ plasmid 3 [15] native array production + Donor DNA CRISPR-Cas9 plasmid Genome editing 2µ plasmid pSNR52 with 5’ HDV Cellobiose utilization 3 [19] + Donor DNA Muconic acid gRNA plasmid + Genome editing Integrated pSNR52 3 [49] production Donor DNA pSNR52 Increase free fatty Genome editing 2µ plasmid tRNA–gRNA CRISPR-Cas9 plasmid 8 [44] acid production array Mevalonate CRISPR-Cas9 plasmid Genome editing CEN plasmid pSNR52 5 [21] production + Donor oligos Leucine and CRISPR-Cas9 plasmid Genome editing 2µ plasmid pSNR52 2 [17] Isoleucine auxotrophy + Donor oligos Genome BDO production; CRISPR-Cas9 plasmid Pathway 2µ plasmid pSNR52 [48] insertion Xylose utilization + Donor DNA insertion Genome CRISPR-Cas9 plasmid 2µ plasmid ptRNA Taxadiene production 4 [23] insertion + Donor DNA Cas9 plasmid + gRNA Genome β-carotene CEN plasmid pSNR52 plasmid + Donor 3 [25] insertion production DNA Genome editing p-coumaric acid Cas9 integration pSNR52 gRNA plasmid 10 [50] and insertion production CRISPR-Cas9 plasmid pSNR52 plus CRISPR Increase ethanol Genome editing 2µ plasmid + Donor DNA 3 [43] native array production plasmid Cas9 plasmid + gRNA Genome editing Enhance synthesis of 2µ plasmid pSNR52 plasmids + Donor 5 [51] and insertion farnesyl diphosphate DNA Cas9 plasmid + gRNA Genome editing Enhance fatty acid 2µ plasmid pSNR52 plasmids + Donor 4 [52] and insertion production DNA Cas9 plasmid + gRNA Genome editing Butanediol 2µ plasmid pSNR52 plasmids + Donor 5 [53] and insertion production DNA Minimize ethyl Genome CRISPR-Cas9 plasmid 2µ plasmid pSNR52 carbamate 1 [54] insertion + accumulation Production of Genome editing gRNA plasmid + Integrated pSNR52 monoterpene 8 [55] and integration Donor DNA precursor, geraniol Cas9 plasmid + gRNA Genome editing CEN plasmid pSNR52 Limonene production plasmid + Donor 9 [56] and integration DNA Genome editing 2-phenylethanol gRNA plasmid + Integrated pSNR52 8 [57] and integration production Donor DNA ARS/CEN plasmid Optimize isoprenoids Gene activation dCas9 plasmid + (dCas9 fused with pSNR52 and triacylglycerols 4 [58] and repression gRNA plasmid VPR domain) biosynthesis ARS/CEN plasmid Cas9 plasmid + Gene activation Optimize α-santalene (Cas9 fused with VPR pSNR52 truncated gRNA 3 [59] and repression biosynthesis domain) plasmid

For genetic engineering purposes, the use of plasmids brings the advantage of expressing a heterologous protein/pathway in a multi-copy way once a 2µ plasmid is maintained at 10–50 copies per cell. However, genome integration has several beneﬁts over plasmids because it allows gene expression stability and lower heterogeneity and eliminates the requirement of using selective growth media after conﬁrmation. Shi et al. [48] developed a CRISPR-based method that allows a multi-integration of a heterologous fragment at the Ty retrotransposon, a set of repetitive sequences (delta (δ) sequences) in the yeast genome (Figure6). The introduction of Cas9-mediated DSBs at δ sites allowed the integration of eighteen copies of a 24 kb DNA fragment carrying a xylose utilization pathway Life 2020, 10, x FOR PEER REVIEW 10 of 17

Nevertheless, some limitations of CRISPR-Cas9 are recognized namely the efficiency Life 2021, 11, 13 variations between different targeted sites [60] and the already mentioned problems 10 of 16 around the polyploid yeast strains. Moreover, the number of multiplex modifications is still limited and some further optimizations are recommended by Zhang et al. [44], namely the identification of stronger RNA polymerase III promoters, the optimization of plasmid constructionand a butanediol tools to production accept multiple pathway. fragments The high with copy repetitive number sequences, of the pathways and resulted the developmentin both of higher new software xylose consumption that correlates and the butanediol gRNA sequences production with by gRNA the engineered effi- strain ciencies. comparatively to the one copy integration one.

Figure 6. Clustered,Figure regularly 6. Clustered interspaced,, regularly short interspaced, palindromic short repeats–associated palindromic repeats Cas9– (CRISPR-Cas9)associated Cas9 associated (CRISPR- with delta Cas9) associated with delta (δ) integration. CRISPR-Cas9 system creates a double-strand break (δ) integration. CRISPR-Cas9 system creates a double-strand break (DSB) at the repetitive δ sequences present in the S. (DSB) at the repetitive δ sequences present in the S. cerevisiae genome allowing the multi-copy cerevisiae genome allowing the multi-copy integration of a DNA fragment containing a gene or even a pathway. integration of a DNA fragment containing a gene or even a pathway. Nevertheless, some limitations of CRISPR-Cas9 are recognized namely the efficiency 5. Innovativevariations CRISPR between Toolkits different in Saccharomyces targeted sites cerevisiae [60] and the already mentioned problems around New emergentthe polyploid procedures yeast strains. into how Moreover, CRISPR the-Cas9 number is applied of multiplex have recently modifications been de- is still limited veloped demonstratingand some further the optimizationspotentialities ofare this recommended tool. The target by Zhang specificity et al. [ 44of], CRISPR namely- the identifi- Cas9 systemscation allowed of stronger researchers RNA polymeraseto develop technologies III promoters, that the take optimization advantage of plasmidof its pre- construction cise DNA targetingtools to accept namely multiple for the fragmentsdelivery of with other repetitive molecules. sequences, EvolvR, for and instance, the development com- of new bines the targetsoftware specificity that correlates of CRISPR the-Cas gRNA9 technology sequences with with the gRNA error efficiencies.-prone capacity of a mutant DNA polymerase for in vivo targeted nucleotide diversification [61]. The system employs the3.3. use Innovative of a nicking CRISPR variant Toolkits of Cas9 in (nCas9) Saccharomyces that cuts cerevisiae only one DNA strand avoiding native homologyNew emergentrepair, and procedures the DNA polymerase into how CRISPR-Cas9 uses the nick is as applied a start havepoint recently to been initiate mutationdeveloped insertion. demonstrating This technology the potentialities was recentl ofy applied this tool. in The S. cerevisiae, target specificity yEvolvR of CRISPR- [62]. The resultsCas9 systemsdemonstrated allowed that researchers yEvolvR towas develop able to technologies insert random that mutations take advantage in both of its precise directions ofDNA the targetingtarget sequence namely, and for the in deliveryaddition, of it otherwas possible molecules. to target EvolvR, two for genomic instance, combines loci at the samethe target time. specificity This technology of CRISPR-Cas9 could be important technology for with fundamental the error-prone eukaryotic capacity re- of a mutant search suchDNA as protein polymerase function for inor vivoproteintargeted interactions nucleotide or to diversification investigate genetic [61]. The mecha- system employs nisms, wherethe yeast use of is ausually nicking used variant as a of role Cas9 model. (nCas9) Moreover that cuts, it only could one be DNA applied strand to strain avoiding native tolerance engineeringhomology repair, for industrial and the DNApurposes. polymerase Engineering uses the yeasts nick to as confer a start them point towith initiate in- mutation creased toleranceinsertion. to an This external technology stress, wassuch recently as temperature applied or in oxidation,S. cerevisiae, is ayEvolvR very valuable [62]. The results strategy todemonstrated improve industrial that yEvolvR strains’ wasperformance. able to insert CRISPR random has mutations already been in both imple- directions of mented forthe random target mutagenesis sequence, and via genome in addition, shuffling. it was Mitsui possible et al. to [63] target applied two genomicCRISPR- loci at the Cas9 for cleavingsame time. the δ- Thissequences technology in order could to fragment be important the chromosome. for fundamental During eukaryotic the repair research such of DNA fragments,as protein large function-scale ormodifications, protein interactions such as orgene to investigateamplification, genetic translocation, mechanisms, where and deletion,yeast may is occur. usually In used this case, as a rolethe DNA model. repair Moreover, was induced it could under be applied thermal to stress strain tolerance conditions.engineering After DNA repair, for industrial the modified purposes. yeastEngineering was able to grow yeasts at to 39 confer °C and them reported with increased higher ethanoltolerance and acid to an resistance external stress,than the such parental as temperature strain. or oxidation, is a very valuable strategy to improve industrial strains’ performance. CRISPR has already been implemented for Targetedrandom regulation mutagenesis of gene expression via genome is shuffling. important Mitsuiboth in et the al. context [63] applied of metabolic CRISPR-Cas9 for δ engineeringcleaving and functional the -sequences genomics in order[34]. In to yeast, fragment the thecontrol chromosome. of genetic During expression the repair is of DNA fragments, large-scale modifications, such as gene amplification, translocation, and deletion, usually performed using characterized gene promoters with different strengths; however may occur. In this case, the DNA repair was induced under thermal stress conditions. After the prediction of the expression level remains a challenge. Qi et al. [64] developed an DNA repair, the modified yeast was able to grow at 39 ◦C and reported higher ethanol and enzymatic version of Cas9 mutated in the nuclease nucleotide sites designated as dead acid resistance than the parental strain. Targeted regulation of gene expression is important both in the context of metabolic engineering and functional genomics [34]. In yeast, the control of genetic expression is

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usually performed using characterized gene promoters with different strengths; however the prediction of the expression level remains a challenge. Qi et al. [64] developed an Life 2020, 10, x FOR PEER REVIEW enzymatic version of Cas9 mutated in the nuclease nucleotide sites designated11 of 17 as dead Cas9 (dCas9). This Cas9 mutant is defective in DNA cleavage, and it can act as a sim- Cas9 (dCas9).ple This specific Cas9 DNA mutant binding is defective complex. in DNA Using cleavage this version, and it of can Cas9 act foras a targeting simple a coding specific DNAsequence binding caused complex. transcriptional Using this version generepression of Cas9 for intargetingEscherichia a coding coli. Thesequence dCas9 binds to caused transcriptionalthe target sequence gene repression blocking in the Escherichia action of RNAcoli. The polymerase. dCas9 binds This to approach the target was named sequence blockingCRISPR interferencethe action of (CRISPRi). RNA polymerase. Next, the This same approach research was group named applied CRISPR the system for interferencegene (CRISPRi). repression Next, in S. thecerevisiae same[ 65 research]. The dCas9 group was applied guided the to a system specific for promoter, gene resulting repression inin anS. cerevisiae efficient gene[65]. The repression. dCas9 was In addition,guided to the a specific repression promoter, can be resulting enhanced in by fusing a an efficienttranscriptional gene repression. repressor In addit domainion, theto dCas9. repression Alternatively, can be enhanced Farzadfard by et al.fusing [66] fused a dCas9 transcriptionalto an repressor activator domain domain and to reported dCas9. Alternatively, an activation or Farzadfard repression et depending al. [66] fused on the targeting dCas9 to ansite. activator When domain the target and was reported outside an theactivation TATA box,or repression the promoter depending was activated on the (CRISPR targeting site.activation When the (CRISPRa)), target was targeting outside adjacentthe TATA to box, the TATAthe promoter box resulted was activated in gene repression (CRISPR activation(CRISPRi) (CRISPRa)), (Figure7). Other targeting activator-domain-fused adjacent to the TATA dCas9 box proteins resulted were in also gene developed repression achieving(CRISPRi) higher (Figure regulation 7). Otherlevels activator [67].-domain Moreover,-fused the dCas9 CRISPRa/i proteins system were has also been applied developed inachieving a polyploid higher yeast regulation strain, which levels can [67] be. very Moreover, valuable the for CRISPRa/i the development system ofhas more robust been appliedindustrial in a polyploid stains [68yeast]. strain, which can be very valuable for the development of more robust industrial stains [68].

Figure 7. Clustered,Figure regularly 7. Clustered interspaced,, regularly short interspaced, palindromic short repeats—interference palindromic repeats— (CRISPRi)interference and (CRISPRi) CRISPR—activation and (CRISPRa) mechanisms.CRISPR— CRISPRiactivation is (CRISPRa) used for gene mechanisms. repression: CRISPRi the dead is used Cas9 for (dCas9) gene repression: with a fused the domaindead Cas9 targets the proximity of TATA(dCas9) box interfering with a fused with domain the formation targets the of theproximity transcriptional of TATA initiation box interfering complex with and the consequently formation repressingof the gene expression.the transcriptional CRISPRa is used initi foration gene complex activation: and dCas9 consequently fused to repressing an activator the domain gene expression. targets outside CRISPRa the vicinity of the TATA box resultingis used for in increasedgene activation: gene expression. dCas9 fused to an activator domain targets outside the vicinity of the TATA box resulting in increased gene expression. Zalatan et al. [69] used a different approach for up/downregulation of a target Zalatangene. et al. Instead [69] used of includinga different fusionapproach domains for up/downregulation in dCas9, the authors of a target included gene. effector pro- Instead of teinincluding recruitment fusion RNAdomains domains in dCas9, into thethe gRNAauthors converting included effector it to a scaffold protein RNA re- (scRNA) cruitment RNA(Figure domains8). The RNAinto the hairpins gRNA of converting scRNA can it recruitto a scaffold a speciﬁc RNA RNA-binding (scRNA) (Figure protein, an acti- 8). The RNAvator hairpins or a repressor, of scRNA thus can used recruit for a locus-speciﬁc specific RNA regulation.-binding protein, Furthermore, an activator Jensen et al. [70] or a repressor,compared thus used the for regulatory locus-specific performance regulation. of two Furthermore, distinct dCas9-mediated Jensen et al. [70] systems: com- using an pared the regulatoryinducible gRNAperformance expression of two and distinct dCas9 dCas9 fused-mediated with a repressor systems: or using an activator an in- domain; ducible gRNAand expression with constitutive and dCas9 expression fused with of scRNAs a repressor for effectoror an activator molecule domain; recruiting. and The two with constitutive expression of scRNAs for effector molecule recruiting. The two systems mediated similar changes in activation/repression of the targeted promoters both at single and at multiplex level.

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systems mediated similar changes in activation/repression of the targeted promoters both at single and at multiplex level. Life 2020, 10, x FOR PEER REVIEW 12 of 17

Figure 8. Mechanism used to associate an effector protein with dead Cas9 (dCas9). The effector molecule sequences can Figure 8. Mechanism used to associate an effector protein with dead Cas9 (dCas9). The effector be fused to the dCas9molecule sequence sequences and can the be fusion fused protein to the dCas9 is expressed sequence in S. and cerevisiae. the fusionOtherwise, protein is endogenous expressed inS. S. cerevisiae effector moleculescerevisiae. can be Otherwise, recruited by en extendingdogenous guideS. cerevisiae RNAseffector to include molecules RNA-based can be effectorrecruited protein by extending recruitment sites (scaffold RNA—scRNA),guide RNAs which to include is advantageous RNA-based for effector repressing protein and recruitment activating multiplesites (scaffold genes RNA simultaneously.—scRNA), CRISPR: clustered, regularlywhich interspaced, is advantageous short palindromic for repressing repeats.and activating multiple genes simultaneously. CRISPR: clustered, regularly interspaced, short palindromic repeats. Moreover, a grade modulation of genetic expression was reported by Deaner and Moreover,Alper a [ 58grade]. The modulation range of genetic of genetic expression expression was relatedwas reported to the proximityby Deaner of and dCas9-based Alper [58]regulators. The range to of the genetic core ofexpression the promoter. was related The grade to the modulation proximity applicabilityof dCas9-based represents a regulatorsstep to the forward core of to the enable promoter. a fine tuningThe grade of metabolic modulation pathways applicability in S. cerevisiaerepresents. The a possible step forwardapplications to enable ofa fine CRISPRi/a tuning of are metabolic massive, pathways and they havein S. cerevisiaealready been. The used possible to improve a applicationsyeast of cellCRISPRi/a factory are to produce massiveβ, -amyrinand they [71 have]. already been used to improve a yeast cell factoryAnother to produce possible β-amyrin approach, [71]. besides the use of a modified CRISPR protein, to guide Anothereffector possible molecules approach, without besides creating the use a ofDSB, a modified is by adjusting CRISPR the protein, gRNA to molecule guide length. effector moleculesTruncated without gRNAs, creating usually a of DSB, 14 nt, is proved by adjusting to be able the to gRNA guide molecule the binding length. of Cas9 to the Truncatedtarget gRNAs, sequence usually without of 14 nt, the proved introduction to be able of to a DSBguide [72 the,73 binding]. Hereupon, of Cas9 truncated to the gRNAs target sequencecan be without used for the transcriptional introduction regulation of a DSB [72, and,73] simultaneously,. Hereupon, truncated full-length gRNAs gRNAs can be can be usedused for fortranscriptional genome editing regulation using aand single, simultaneously Cas9 protein., full This-length feature gRNAs allowed can researchers be to used for genomedevelopediting multifunctional using a single systems Cas9 capable protein. of one-pot This feature CRISPRi, allowed CRISPRa, researchers or CRISPR to editing. develop multifunctionalLian et al. [74] reportedsystems capable the first of trifunctional one-pot CRISPRi, CRISPR CRISPRa system for, or simultaneousCRISPR edit- gene inac- ing. Lian ettivation, al. [74] activation,reported the and first editing trifunctional in S. cerevisiae CRISPRusing system truncated for simultaneous gRNAs for gene CRISPRa and inactivation,CRISPRi, activation called, and CRISPR-AID. editing in S. However, cerevisiae to using avoid truncated competition gRNAs between for gRNAsCRISPRa for the same and CRISPRi,Cas9 called protein, CRISPR three-AID. different However, PAM-recognizing to avoid competition CRISPR proteins between were gRNAs used. for More the recently, same Cas9Dong protein, et al. three [59] differentestablished PAM a trifunctional-recognizing CRISPR CRISPR systemproteins using were aused. single More Cas9 protein. The system was named CRISPR-ARE and employed the use of a Cas9 fused to a VP64-p65- recently, Dong et al. [59] established a trifunctional CRISPR system using a single Cas9 Rta (VPR) activation domain. The authors demonstrated the applicability of CRISPR-ARE protein. The system was named CRISPR-ARE and employed the use of a Cas9 fused to a by optimizing α-santalene biosynthesis. The system used truncated gRNAs to target the VP64-p65-Rta (VPR) activation domain. The authors demonstrated the applicability of genes for activation and repression and full-length gRNAs to edit one gene by transform- CRISPR-ARE by optimizing α-santalene biosynthesis. The system used truncated gRNAs ing together the donor repair DNA. The editing efficiency was 100% and, gene activation to target the genes for activation and repression and full-length gRNAs to edit one gene and gene repression were confirmed using a reporter protein. Regarding the α-santalene by transforming together the donor repair DNA. The editing efficiency was 100% and, biosynthesis, it increased 2.66-fold. gene activation and gene repression were confirmed using a reporter protein. Regarding the α-santalene biosynthesis, it increased 2.66-fold.

6. Conclusions In summary, researchers have been taking advantage of the specificity of the CRISPR- Cas9 system, not only to perform direct genetic modifications but also to direct other molecules (activators or DNA polymerases) to a previously defined locus. Engineering Cas9 protein (dCas9 or nCas9) allowed the targeting of specific sequences without introducing Life 2021, 11, 13 13 of 16

4. Conclusions In summary, researchers have been taking advantage of the specificity of the CRISPR- Cas9 system, not only to perform direct genetic modifications but also to direct other molecules (activators or DNA polymerases) to a previously defined locus. Engineering Cas9 protein (dCas9 or nCas9) allowed the targeting of specific sequences without introducing a DSB, which can be used to guide these molecules. Moreover, the use of truncated gRNAs allowed the development of multifunctional CRISPR systems, such CRISPR-AID and CRISPR-ARE, which will facilitate fast and multifunctional engineering of the S. cerevisiae chassis. Key Messages: The CRISPR-Cas9 system’s discovery is with no doubt a landmark in the synthetic biology field. The simplicity and flexibility of the system enabled the researchers to develop a panoply of CRISPR applications. Consequently, it is now possible to build more and more complex designs in yeast by multiplexed CRISPR-Cas9 genome editing. Besides allowing multiple modifications, the emergence of CRISPR-Cas9 enabled the researchers to perform genetic modifications faster, cheaper, and more efficiently. The possibility to perform genetic modifications without the requirement of selective markers or the multiplex genomic edits are examples of the possibilities brought by this technology. The scientific community is taking advantage of the fantastic teamwork between CRISPR and yeast with results ranging from the introduction of large chromosomal segments to point mutations and by developing improved genome editing systems. Moreover, CRISPR applications have transgressed the conventional genome editing application through the creation of novel mutated versions of Cas9 such as nCas9 or dCas9. CRISPRi/a is a tremendously useful tool for transcriptional regulation because it allows to balance and optimize gene expression without genome editing. This tool may be tremendously useful when dealing with essential genes in S. cerevisiae mainly for the development of industrial strains where the metabolic fluxes need to be meticulously balanced. The application of multifunctional systems, such as CRISPR-ARE, accelerates the design–build–test processes and consequently the productivity of the research. The EvolvR technology may facilitate the development of highly adapted S. cerevisiae strains or even simplify the study of the eukaryotic genetic mechanisms. Finally, we believe that various applications of the CRISPR- Cas9 systems in S. cerevisiae will continue to evolve in order to respond to the challenges of the biotechnological field and to contribute to the sustainability of the bio-based industry.

Author Contributions: J.R. performed the literature review and drafted the manuscript. J.L.R. and L.R.R. assisted in the manuscript conception and design and provided feedback and suggestions on the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020—North Portugal Regional Program. In addition, this research has been carried out at the Biomass and Bioenergy Research Infrastructure (BBRI)—LISBOA-010145- FEDER-022059, supported by Operational Program for Competitiveness and Internationalization (PORTUGAL2020), the Lisbon Portugal Regional Operational Program (Lisboa2020), and Norte2020 under the Portugal 2020 Partnership Agreement, through the ERDF. J.R. is recipient of a fellowship supported by a doctoral advanced training (SFRH/BD/138325/2018) funded by FCT. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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