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. 2016 Jul 7;6(7):2147-56.
doi: 10.1534/g3.116.029801.

mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence

Gregory C Finnigan  1 , Jeremy Thorner  2
Affiliations

mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence

Gregory C Finnigan et al. G3 (Bethesda). .

Erratum in

  • Corrigendum.
    [No authors listed] [No authors listed] G3 (Bethesda). 2019 Apr 9;9(4):1279. doi: 10.1534/g3.119.400096. G3 (Bethesda). 2019. PMID: 30967430 Free PMC article. No abstract available.

Abstract

Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly) manipulated at multiple loci with extremely high efficiency.

Keywords: CRISPR; Saccharomyces cerevisiae; essential genes; genome editing; genome engineering.

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Figures

Figure 1
Figure 1
Installation of programmed non-yeast Cas9 target sites at multiple loci. (A) Haploid yeast strains were constructed in which the endogenous CDC11 gene and a shs1∆::HygR allele were flanked by an identical 23 bp sequence containing a Cas9 target site (including a 5′-NGG-3′ PAM sequence) from the human SEPT9 gene, designated “unique Cas9 site 1,” u1. At CDC11, the upstream u1 site was placed in-frame with the initiator Met of the ORF, and the downstream u1 was kept in-frame with the stop codon (via addition of an A to the 5′-end of each u1). Because CDC11 is an essential gene, a URA3-marked CEN plasmid expressing WT CDC11 (but with no 3′-UTR) was also present. Red triangles, site of Cas9-directed DSB (+ 3 upstream of the PAM). (B) A cassette for inducible GAL1/10 promoter-driven expression of S.p.Cas9 bearing a C-terminal SV40 NLS and a ADH1 transcriptional terminator was used to replace the ORF at the endogenous HIS3 locus. In one variant (strain GFY-2002), this cassette was flanked by u2, a different 23 bp human sequence containing a Cas9 target from the human MMP23A locus. In another variant (strain GFY-2003), the cassette was flanked by u1. (C) The corresponding sgRNA[u1] and sgRNA[u2] sequences were expressed using the constitutive yeast pol III snoRNA SNR52 promoter and yeast pol III tRNA SUP4 terminator on high-copy (2 μm DNA) plasmids. DSB, double-strand break; HygR, hygromycin resistance; KanR, kanamycin resistance; NLS, nuclear localization signal; ORF, open reading frame; PAM, protospacer adjacent motif; sgRNA, single-guide RNA; tRNA, transfer RNA; UTR, untranslated region; WT, wild-type.
Figure 2
Figure 2
Multiplex Cas9-mediated scarless gene replacement (including an essential gene) and optional concurrent elimination of Cas9. (A) Otherwise isogenic yeast strains containing six programmed Cas9 target sites. In strain GFY-2002, the CDC11 and shs1∆::HygR loci are flanked by u1, whereas the Cas9 expression cassette at the HIS3 locus is flanked by u2. In strain GFY-2003, all three loci are flanked by u1. Both strains also carried a URA3-marked CEN plasmid harboring WT CDC11. (B) Cas9 expression was induced in strains GFY-2002 (left) or GFY-2003 (right), and then the cells were transformed with an empty LEU2-marked vector (pRS425) or with the same plasmid expressing sgRNA[u1] in the absence or presence of various combinations of PCR fragments that span each of the genomic loci of interest, as indicated. The PCR fragments contained either 500 bp (upper plates) or just 30 bp (lower plates) of homology to the genomic sequence flanking each locus. Asterisk, for the CDC11 PCR fragment, the flanking homology was 330 bp. After recovery in rich medium containing galactose (to support continued Cas9 expression), the cells were plated on SD-Ura-Leu medium. The plates were imaged and the number of colonies recovered were counted after incubation at 30° for 3 d. Each independent trial was performed in triplicate. Representative plates are shown; white numbers, total colony count. The empty vector control confirmed that these conditions allowed for efficient transformation and selection for the LEU2- and URA3-marked plasmids. Individual colonies from Condition A, where all of the PCR fragments necessary to heal the Cas9-sgRNA[u1]-generated DSBs were provided, were tested for growth on various diagnostic media to ascertain whether successful gene replacement occurred (see Table S3). Red values, percentage of colonies scored that exhibited successful gene replacement at all loci tested. (C) The average colony count over all experimental trials for each condition (A–D), as indicated. Error bars, SEM. (D) An isolate of GFY-2002 from Condition A (B and C) in which both the u1-flanked CDC11 locus and u1-flanked shs1∆::HygR allele were successfully replaced with WT CDC11 (see Figure 3) and WT SHS1, respectively, was grown in galactose to induce Cas9 expression, and then transformed with either empty vector (pRS423) or the same plasmid expressing sgRNA[u2], plated on SD-Ura-His medium, and grown at 30° for 3 d. The selectable marker in the sgRNA[u2]-expressing plasmid is the S. cerevisiae HIS3 gene with 317 bp of 5′- and 201 bp of 3′-flanking genomic sequence. Therefore, this plasmid not only provides sgRNA[u2] to target Cas9 cleavage at the u2 sites flanking the his3∆::Cas9::KanR cassette, but it also serves as a source of WT HIS3 DNA to repair the cleaved locus. Representative plates are shown; white numbers, total colony count. To assess conversion of the u2-flanked his3∆::Cas9::KanR cassette to WT HIS3, the His+ Ura+ colonies obtained were scored for loss of G418 resistance and complete elimination of the entire cassette (Table S4). Red values, percentage of colonies scored that exhibited successful elimination of the his3∆::Cas9::KanR cassette. HygR, hygromycin resistance; KanR, kanamycin resistance; ORF, open reading frame; PCR, polymerase chain reaction; sgRNA, single-guide RNA; UTR, untranslated region; WT, wild-type.
Figure 3
Figure 3
Diagnostic PCR confirms efficient multiplex gene replacement. (A) Chromosomal DNA was purified (Amberg et al. 2006) from 10, randomly chosen, clonal isolates from transformations of GFY-2002 in which PCR fragments with 500 bp of flanking genomic homology were provided to restore WT CDC11 and WT SHS1 loci, and which had lost the HygR marker (see Figure 2B), and tested by PCR with the indicated diagnostic primer sets. An identical analysis was performed on 10 isolates in which PCR fragments with only 30 bp of flanking genomic homology were provided and which had lost the HygR marker (see Figure S3). The PCR products were resolved by agarose gel electrophoresis and visualized by staining with ethidium bromide. For CDC11 (top three gels), the entire locus was amplified (primers F1/R1), as well as small fragments flanking the upstream (F2/R2) or downstream (F3/R3) u1 sites to determine whether or not the Cas9 target site was still present. For SHS1 (fourth and fifth gels), PCR was performed using primers unique to either SHS1 itself (F4/R4) or to the HygR cassette (F4/R5). Finally, the HIS3 locus (bottom gel) was testing using a unique primer internal to the Cas9 gene and to the KanR cassette (F5/R6). For optimal separation, 2% agarose was used for the second and third gels, 1% agarose was used for all of the others. Left, nearest DNA size marker (in kb) for each independent gel; right, expected PCR product sizes. (B) The same kind of analysis as in (A) was performed on chromosomal DNA purified from 10, randomly chosen, clonal isolates from transformations of GFY-2003, except that, in addition, PCR diagnostic for the HIS3 locus was performed (F6/R7) to ascertain whether the u1-flanked his3∆::Cas9::KanR cassette had been replaced by the WT HIS3 gene. Left red asterisks, three representative isolates of GFY-2002 that diagnostic PCR indicated carried WT CDC11 and WT SHS1 loci, and retained the u2-flanked his3∆::Cas9::KanR cassette, were confirmed as such by direct DNA sequencing (data not shown). Right red asterisks, three representative isolates of GFY-2003 that diagnostic PCR indicated carried WT CDC11, WT SHS1, and WT HIS3 loci, were also confirmed as such by direct DNA sequencing (data not shown) [for diagnostic PCR and sequencing of surviving colonies from controls (Figure 2B, conditions B–D), see Figure S5]. HygR, hygromycin resistance; KanR, kanamycin resistance; ORF, open reading frame; PCR, polymerase chain reaction; sgRNA, single-guide RNA; UTR, untranslated region; WT, wild-type.
Figure 4
Figure 4
Comparison of Cas9-mediated genome editing by multiplexing sgRNAs vs. multiplexing loci with a unique target site. (A) Traditional targeting of Cas9 to multiple genomic loci (including one locus where Cas9 is integrated). Each of four loci is illustrated as requiring Cas9 action at two distinct sites. Hence, concurrent action of Cas9 at these four genes would require the selection of eight individual PAM-containing genomic sequences and the production of eight corresponding sgRNAs. In addition, it should be noted that, in this scenario, at least one target site lies within the coding sequence of each gene; therefore, PCR fragments used to replace Genes(1–3) would also require alterations of the coding sequence to avoid recutting by Cas9 (also see Figure S1). Finally, for manipulation of any essential genes (e.g., Gene1), a counterselectable plasmid expressing a WT copy will also need to be altered to not include the genomic target site(s), again to avoid its Cas9-mediated cleavage (Figure S1). (B) The approach of multiplexing the target site(s) has a number of useful advantages. First, there is no need to restrict the target for Cas9 cleavage to sequences that exist within the genome of interest, which may be suboptimal (with regard to off-target effects) or may have a limited number or inopportune placements of available PAM sites. Second, the artificial target site chosen for insertion may be any stretch of 23 nucleotides (20 plus a 5′-NGG-3′ PAM) taken from any known species (or designed de novo), as long as it has no counterpart in the genome of interest. In fact, such a programmed target site sequence should greatly reduce or eliminate off-target effects, and also has the virtue that it can be inserted at a precise location (down to the base pair) to optimally facilitate recombination and precisely control the placement of the Cas9-mediated DSBs. The limiting step in this approach is, of course, introduction of these unique target site insertions into the parental genome at the desired locations. Once created, however, such an engineered parental strain can be used repeatedly to install various different alterations at one or many loci using only a single sgRNA, allowing for rapid construction of multiple strain variants. Moreover, in this approach, the Cas9 expression cassette can be retained, targeted for simultaneous excision in parallel with the manipulations of other loci (right), or eliminated at a later time, if the Cas9 expression cassette is flanked with a separate unique target site (left). Finally, because the sequence of the target sites flanking each locus are distinct from any of the elements of the targeted genes themselves, no modifications to the sequence of the PCR fragments used for gene editing (or of a covering plasmid carrying the corresponding WT gene) are required to make them immune to the further action of Cas9. DSB, double-strand break; PAM, protospacer adjacent motif; PCR, polymerase chain reaction; sgRNA, single-guide RNA; UTR, untranslated region; WT, wild-type.
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