Highly Parallel Gene Synthesis
The commercial cost of large-scale DNA synthesis makes it prohibitively expensive to make millions of genome-scale constructions as required for optimization of metabolic and genetic systems. To overcome this limiting factor, the team led by Dr. Church utilizes an unorthodox gene-synthesis method, which he claims reduces the cost at least 20-fold. First, a computer algorithm helps to break down the desired sequence into 40–150 bp pieces. Then these oligonucleotides are printed on custom microchips, which can pack as many as 55,000 individual sequences.
“The advantage of this method is in highly parallel synthesis of thousands of DNA fragments,” says Emily Leproust, Ph.D., director, applications and chemistry R&D, Agilent Technologies. “Any conceivable DNA variants can be all done at the same time. The resulting pool of oligos can serve as a starting point for many creative applications.”
Dr. Church’s team amplified the oligo pool, generated from Agilent’s Oligo Library Synthesis (OLS) platform, via several rounds of PCR reactions. The fragments were linked together, creating hundreds of genetic products of kilobases or more.
“This technique is a core technology at the Center for Causal Consequences of Variation,” comments Dr. Church. “We continue the optimization of the process in order to make writing of human genome accurate and efficient.” Writing, or editing, of the genome on the large scale will enable researchers to test what variations in the human genome really cause diseases.
“By engineering many combinations of such variations, and directly observing their impact on the cell, we will be able to establish cause and effect in a more direct way than genome-wide association studies allow for.”
“To test the genetic variations, we place our synthetic constructs into human cells by using synthetic proteins.” The team has generated artificial versions of TALEs (transcription activator-like effectors), DNA-binding proteins found in the plant pathogen Xanthomonas spp. Each monomeric domain of a TALE can be engineered to recognize just one specific nucleotide. A specific arrangement of TALE monomers, therefore, will recognize a specific DNA motif of 18–30 base pairs.
TALEs bring with them the enzymes for DNA cleavage and recombination, enabling the insertion of the synthetic DNA precisely in the desired location.
Creatively designed OLS pools have been used in generating synthetic bioblocks. Researchers from the University of California, San Francisco designed OLS microchips containing thousands of short hairpin RNAs (shRNAs). At approximately 30 shRNA/gene, the pool targeted ~600 genes.
“Without Agilent OLS technology, this would be cost-prohibitive,” adds Dr. Leproust. “We print oligos on the femtomole scale, >1,000 times lower output than the column-based method. And the library now can be created without the extensive cost of synthesizing and assaying the silencing efficacy of these oligos one at a time, but rather as a pool in a simple tube assay.”
The shRNA library is delivered into cells by lentiviruses. The outcome is followed by sorting of individual cells according to a desired phenotype. OLS strategy was also used to accelerate discovery of new bioblocks such as promoter elements. Synthetic promoters containing every possible combination of mutations were printed on the array and assayed as a pool off the chip for transcriptional efficiencies.
In a single experiment, researchers assayed the effects of all possible single-nucleotide mutations in a given promoter region. “Saturation mutagenesis on this scale is simply impossible by other methods,” concludes Dr. Leproust.