scholarly journals Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Satoshi Okada ◽  
Goro Doi ◽  
Shitomi Nakagawa ◽  
Emiko Kusumoto ◽  
Takashi Ito
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Editing ◽  
Region Of Interest ◽  
Single Amino Acid ◽  
Yeast Genome ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Protospacer Adjacent Motif ◽  
Combined Use ◽  
Genome Manipulation

Abstract Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.

scholarly journals Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Satoshi Okada ◽  
Goro Doi ◽  
Shitomi Nakagawa ◽  
Emiko Kusumoto ◽  
Takashi Ito
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Editing ◽  
Region Of Interest ◽  
Yeast Genome ◽  
Yeast Genetics ◽  
Reading Frame ◽  
Protospacer Adjacent Motif ◽  
Combined Use ◽  
Genome Manipulation ◽  
Complete Deletion

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing and their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a software to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in both insertion of gene fragments to essential genes and complete deletion of an open reading frame. The backbone vectors with the software would thus provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1

1988 ◽  
Vol 8 (4) ◽  
pp. 1432-1442 ◽  
Author(s):  
J D Boeke ◽  
D Eichinger ◽  
D Castrillon ◽  
G R Fink
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Long Terminal Repeat ◽  
Missense Mutation ◽  
Dna Sequence ◽  
Open Reading Frame ◽  
Yeast Genome ◽  
Reading Frame ◽  
Ty Elements ◽  
Ty Element ◽  
Repeat Sequences

Saccharomyces cerevisiae Ty elements are transposons closely related to retroviruses. The DNA sequence of a functional Ty element (TyH3) is presented. The long terminal repeat sequences are different, suggesting that TyH3 is a recombinant Ty element. A chromosomal Ty element near the LYS2 gene, Ty173, was found to be nonfunctional, even though it has no detectable insertions or deletions. The defect in Ty173 transposition is caused by a missense mutation giving rise to a Leu-to-Ile substitution in the TYB (pol) open reading frame. Several chromosomal Ty elements carry this lesion in their DNA, indicating that nonfunctional Ty elements are common in the yeast genome.

Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae

1989 ◽  
Vol 9 (9) ◽  
pp. 3638-3646
Author(s):  
C M Nicolet ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Saccharomyces Cerevisiae ◽  
Two Dimensional Gel Electrophoresis ◽  
Reading Frame ◽  
Hsp70 Family ◽  
Isolation And Characterization ◽  
Isoelectric Points ◽  
Amino Acid Analog ◽  
Inducible Gene

We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a 2.0-kilobase heat-inducible mRNA. This gene, which we have designated STI1, for stress inducible, was also induced by the amino acid analog canavanine and showed a slight increase in expression as cells moved into stationary phase. The STI1 gene encodes a 66-kilodalton protein, as determined from the sequence of the longest open reading frame. The putative STI1 protein, as identified by two-dimensional gel electrophoresis, migrated in the region of 73 to 75 kilodaltons as a series of four isoforms with different isoelectric points. STI1 is not homologous to the other conserved HSP70 family members in yeasts, despite similarities in size and regulation. Cells carrying a disruption mutation of the STI1 gene grew normally at 30 degrees C but showed impaired growth at higher and lower temperatures. Overexpression of the STI1 gene resulted in substantial trans-activation of SSA4 promoter-reporter gene fusions, indicating that STI1 may play a role in mediating the heat shock response of some HSP70 genes.

scholarly journals Two Saccharomyces cerevisiae genes which control sensitivity to G1 arrest induced by Kluyveromyces lactis toxin.

1994 ◽  
Vol 14 (9) ◽  
pp. 6306-6316 ◽  
Author(s):  
A R Butler ◽  
J H White ◽  
Y Folawiyo ◽  
A Edlin ◽  
D Gardiner ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Copy Number ◽  
Kluyveromyces Lactis ◽  
Yeast Cells ◽  
Yeast Genome ◽  
G1 Arrest ◽  
Yeast Saccharomyces Cerevisiae ◽  
Toxin Resistance ◽  
Complementation Groups ◽  
Diploid Cells

The Kluyveromyces lactis toxin causes an arrest of sensitive yeast cells in the G1 phase of the cell division cycle. Two complementary genetic approaches have been undertaken in the yeast Saccharomyces cerevisiae to understand the mode of action of this toxin. First, two sequences conferring toxin resistance specifically in high copy number have been isolated and shown to encode a tRNA(Glu3) and a novel polypeptide. Disruption of the latter sequence in the yeast genome conferred toxin resistance and revealed that it was nonessential, while the effect of the tRNA(Glu)3 was highly specific and mediated resistance by affecting the toxin's target. An alpha-specific, copy number-independent suppressor of toxin sensitivity was also isolated and identified as MATa, consistent with the observation that diploid cells are partially resistant to the toxin. Second, in a comprehensive screen for toxin-resistant mutants, representatives of 13 complementation groups have been obtained and characterized to determine whether they are altered in the toxin's intracellular target. Of 10 genes found to affect the target process, one (KTI12) was found to encode the novel polypeptide previously identified as a multicopy resistance determinant. Thus, both loss of KTI12 function and elevated KTI12 copy number can cause resistance to the K. lactis toxin.

scholarly journals The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1.

1988 ◽  
Vol 8 (4) ◽  
pp. 1432-1442 ◽  
Author(s):  
J D Boeke ◽  
D Eichinger ◽  
D Castrillon ◽  
G R Fink
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Long Terminal Repeat ◽  
Missense Mutation ◽  
Dna Sequence ◽  
Open Reading Frame ◽  
Yeast Genome ◽  
Reading Frame ◽  
Ty Elements ◽  
Ty Element ◽  
Repeat Sequences

Saccharomyces cerevisiae Ty elements are transposons closely related to retroviruses. The DNA sequence of a functional Ty element (TyH3) is presented. The long terminal repeat sequences are different, suggesting that TyH3 is a recombinant Ty element. A chromosomal Ty element near the LYS2 gene, Ty173, was found to be nonfunctional, even though it has no detectable insertions or deletions. The defect in Ty173 transposition is caused by a missense mutation giving rise to a Leu-to-Ile substitution in the TYB (pol) open reading frame. Several chromosomal Ty elements carry this lesion in their DNA, indicating that nonfunctional Ty elements are common in the yeast genome.

scholarly journals Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

1994 ◽  
Vol 5 (9) ◽  
pp. 933-942 ◽  
Author(s):  
H Fang ◽  
N Green
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Water Soluble ◽  
Growth Defect ◽  
Sequence Analyses ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Transmembrane Glycoprotein ◽  
Genetic Assay ◽  
Er Membrane

The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane.

scholarly journals Deletion of a single-copy tRNA affects microtubule function in Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (4) ◽  
pp. 955-962 ◽  
Author(s):  
R A Reijo ◽  
D S Cho ◽  
T C Huffaker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Copy ◽  
Open Reading Frame ◽  
Northern Analysis ◽  
Trna Genes ◽  
Heat Sensitivity ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Sensitive Allele

Abstract rts1-1 was identified as an extragenic suppressor of tub2-104, a cold-sensitive allele of the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. In addition, rts1-1 cells are heat sensitive and resistant to the microtubule-destabilizing drug, benomyl. The rts1-1 mutation is a deletion of approximately 5 kb of genomic DNA on chromosome X that includes one open reading frame and three tRNA genes. Dissection of this region shows that heat sensitivity is due to deletion of the open reading frame (HIT1). Suppression and benomyl resistance are caused by deletion of the gene encoding a tRNA(Arg)AGG (HSX1). Northern analysis of rts1-1 cells indicates that HSX1 is the only gene encoding this tRNA. Deletion of HSX1 does not suppress the tub2-104 mutation by misreading at the AGG codons in TUB2. It also does not suppress by interfering with the protein arginylation that targets certain proteins for degradation. These results leave open the prospect that this tRNA(Arg)AGG plays a novel role in the cell.

scholarly journals Sequencing and functional analysis of the yeast genome.

1998 ◽  
Vol 45 (3) ◽  
pp. 627-643 ◽  
Author(s):  
M Zagulski ◽  
C J Herbert ◽  
J Rytka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Eukaryotic Genome ◽  
Model Organisms ◽  
Yeast Genome ◽  
Entire Genome ◽  
Yeast Saccharomyces Cerevisiae ◽  
Entire Genome Sequence ◽  
Sequencing Project ◽  
New Genes ◽  
Public Data

The genome of the yeast Saccharomyces cerevisiae was sequenced by an international consortium of laboratories from Europe, Canada, the U.S.A. and Japan. This project is now finished and the complete sequence of the first eukaryotic genome was released to the public data bases in April 1996. An overview and preliminary analysis of the entire genome sequence was presented in a special issue of Nature in May 1997, entitled "The yeast genome directory". At its origin the Yeast Genome Sequencing Project provoked much debate and controversy; however, the final results obtained and the insights this has given us into the organisation and content of a eukaryotic genome have more than justified the expectations of the supporters of the project. The importance of genomic sequencing and analysis, especially of model organisms, is now widely accepted and this has resulted in the birth of the new science of genomics (Botstein & Cherry, 1997, Proc. Natl. Acad. Sci. U.S.A. 94, 5506). The information from gene and protein sequences ultimately lead to functional description of all genes. The main strategies describing possible ways to analyse the function of new genes that have been identified by systematic sequencing of Saccharomyces cerevisiae genome are described.

scholarly journals Rearrangements occurring adjacent to a single Ty1 yeast retrotransposon in the presence and absence of full-length Ty1 transcription.

Genetics ◽  
1992 ◽  
Vol 131 (4) ◽  
pp. 833-850
Author(s):  
P R Sutton ◽  
S W Liebman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Transposable Element ◽  
Gene Conversion ◽  
Genetic Background ◽  
Repetitive Sequences ◽  
Full Length ◽  
Yeast Genome ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ty1 Retrotransposon

Abstract The structures of two unusual deletions from the yeast Saccharomyces cerevisiae are described. These deletions extend from a single Ty1 retrotransposon to an endpoint near a repetitive tRNA(Gly) gene. The deletions suggest that unique sequences flanked by two nonidentical repetitive sequences, or bordered on only one side by a transposable element, have the potential to be mobilized in the yeast genome. Models for the formation of these two unusual deletions were tested by isolating and analyzing 32 additional unusual deletions of the CYC1 region that extend from a single Ty1 retrotransposon. Unlike the most common class of deletions recovered in this region, these deletions are not attributable solely to homologous recombination among repetitive Ty1 or delta elements. They arose by two distinct mechanisms. In an SPT8 genetic background, most unusual deletions arose by transposition of a Ty1 element to a position adjacent to a tRNA(Gly) gene followed by Ty1-Ty1 recombination. In an spt8 strain, where full-length Ty1 transcription and, therefore, transposition are reduced, most deletions were due to gene conversion of a 7-kb chromosomal interval flanked by a Ty1 element and a tRNA(Gly) gene.

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