PMID- 25539750
OWN - NLM
STAT- MEDLINE
DCOM- 20150807
LR  - 20181113
IS  - 1471-2164 (Electronic)
IS  - 1471-2164 (Linking)
VI  - 15
IP  - 1
DP  - 2014 Dec 24
TI  - TREC-IN: gene knock-in genetic tool for genomes cloned in yeast.
PG  - 1180
LID - 10.1186/1471-2164-15-1180 [doi]
LID - 1180
AB  - BACKGROUND: With the development of several new technologies using synthetic 
      biology, it is possible to engineer genetically intractable organisms including 
      Mycoplasma mycoides subspecies capri (Mmc), by cloning the intact bacterial 
      genome in yeast, using the host yeast's genetic tools to modify the cloned 
      genome, and subsequently transplanting the modified genome into a recipient cell 
      to obtain mutant cells encoded by the modified genome. The recently described 
      tandem repeat coupled with endonuclease cleavage (TREC) method has been 
      successfully used to generate seamless deletions and point mutations in the 
      mycoplasma genome using the yeast DNA repair machinery. But, attempts to knock-in 
      genes in some cases have encountered a high background of transformation due to 
      maintenance of unwanted circularization of the transforming DNA, which contains 
      possible autonomously replicating sequence (ARS) activity. To overcome this 
      issue, we incorporated a split marker system into the TREC method, enabling 
      seamless gene knock-in with high efficiency. The modified method is called 
      TREC-assisted gene knock-in (TREC-IN). Since a gene to be knocked-in is delivered 
      by a truncated non-functional marker, the background caused by an incomplete 
      integration is essentially eliminated. RESULTS: In this paper, we demonstrate 
      applications of the TREC-IN method in gene complementation and genome 
      minimization studies in Mmc. In the first example, the Mmc dnaA gene was 
      seamlessly replaced by an orthologous gene, which shares a high degree of 
      identity at the nucleotide level with the original Mmc gene, with high efficiency 
      and low background. In the minimization example, we replaced an essential gene 
      back into the genome that was present in the middle of a cluster of non-essential 
      genes, while deleting the non-essential gene cluster, again with low backgrounds 
      of transformation and high efficiency. CONCLUSION: Although we have demonstrated 
      the feasibility of TREC-IN in gene complementation and genome minimization 
      studies in Mmc, the applicability of TREC-IN ranges widely. This method proves to 
      be a valuable genetic tool that can be extended for genomic engineering in other 
      genetically intractable organisms, where it may be implemented in elucidating 
      specific metabolic pathways and in rationale vaccine design.
FAU - Chandran, Suchismita
AU  - Chandran S
FAU - Noskov, Vladimir N
AU  - Noskov VN
FAU - Segall-Shapiro, Thomas H
AU  - Segall-Shapiro TH
FAU - Ma, Li
AU  - Ma L
FAU - Whiteis, Caitlin
AU  - Whiteis C
FAU - Lartigue, Carole
AU  - Lartigue C
FAU - Jores, Joerg
AU  - Jores J
FAU - Vashee, Sanjay
AU  - Vashee S
FAU - Chuang, Ray-Yuan
AU  - Chuang RY
AD  - The J, Craig Venter Institute, 9704 Medical Center Drive, Rockville 20850, MD, 
      USA. rchuang@jcvi.org.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20141224
PL  - England
TA  - BMC Genomics
JT  - BMC genomics
JID - 100965258
SB  - IM
MH  - *Cloning, Molecular/methods
MH  - *Gene Knock-In Techniques
MH  - Gene Order
MH  - Genes, Fungal
MH  - Genetic Vectors/genetics
MH  - *Genome, Fungal
MH  - *Genomics/methods
MH  - Mycoplasma mycoides/genetics
MH  - Saccharomyces cerevisiae/genetics
MH  - Yeasts/*genetics
PMC - PMC4407568
EDAT- 2014/12/30 06:00
MHDA- 2015/08/08 06:00
PMCR- 2014/12/24
CRDT- 2014/12/26 06:00
PHST- 2014/10/18 00:00 [received]
PHST- 2014/12/12 00:00 [accepted]
PHST- 2014/12/26 06:00 [entrez]
PHST- 2014/12/30 06:00 [pubmed]
PHST- 2015/08/08 06:00 [medline]
PHST- 2014/12/24 00:00 [pmc-release]
AID - 1471-2164-15-1180 [pii]
AID - 6985 [pii]
AID - 10.1186/1471-2164-15-1180 [doi]
PST - epublish
SO  - BMC Genomics. 2014 Dec 24;15(1):1180. doi: 10.1186/1471-2164-15-1180.