Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.

[1]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.

[2]  Feng Zhang,et al.  CRISPR-assisted editing of bacterial genomes , 2013, Nature Biotechnology.

[3]  J. Acker,et al.  Yeast RNA polymerase III transcription factors and effectors. , 2013, Biochimica et Biophysica Acta.

[4]  Jeffry D. Sander,et al.  Efficient In Vivo Genome Editing Using RNA-Guided Nucleases , 2013, Nature Biotechnology.

[5]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[6]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[7]  Jennifer Doudna,et al.  RNA-programmed genome editing in human cells , 2013, eLife.

[8]  Dana Carroll,et al.  A CRISPR approach to gene targeting. , 2012, Molecular therapy : the journal of the American Society of Gene Therapy.

[9]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[10]  Lei Wang,et al.  Genetic incorporation of unnatural amino acids into proteins in yeast. , 2012, Methods in molecular biology.

[11]  R. Barrangou,et al.  CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. , 2011, Annual review of genetics.

[12]  J. Vogel,et al.  CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III , 2011, Nature.

[13]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[14]  Qian Wang,et al.  New methods enabling efficient incorporation of unnatural amino acids in yeast. , 2008, Journal of the American Chemical Society.

[15]  D. Gordenin,et al.  Conservative Repair of a Chromosomal Double-Strand Break by Single-Strand DNA through Two Steps of Annealing , 2006, Molecular and Cellular Biology.

[16]  Riccardo Percudani,et al.  Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III* , 2005, Journal of Biological Chemistry.

[17]  R. Schiestl,et al.  High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier , 1989, Current Genetics.

[18]  D. Gordenin,et al.  Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  B. Shafer,et al.  Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1. , 2001, Genetics.

[20]  T G Burland,et al.  DNASTAR's Lasergene sequence analysis software. , 2000, Methods in molecular biology.

[21]  M. Resnick,et al.  A double-strand break within a yeast artificial chromosome (YAC) containing human DNA can result in YAC loss, deletion or cell lethality , 1996, Molecular and cellular biology.

[22]  R. Müller,et al.  Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. , 1994, Nucleic acids research.

[23]  J. K. Bhattacharjee,et al.  Genetic and biochemical properties of thialysine-resistant mutants of Saccharomyces cerevisiae. , 1981, Journal of general microbiology.

[24]  T. Manney,et al.  The CAN1 locus of Saccharomyces cerevisiae: fine-structure analysis and forward mutation rates. , 1979, Genetics.