Efficient and Low-Cost Error Removal in DNA Synthesis by a High-Durability MutS

Enzyme-based error correction is
a key step in de novo DNA synthesis, yet the inherent
instability of error-correction
enzymes such as MutS has hindered the throughput and efficiency of
DNA synthesis workflows. Here we introduce a process called Improved
MICC (iMICC), in which all error-correction steps of oligos and fragments
within a complete gene-synthesis cycle are completed in a simple,
efficient, and low-cost manner via a MutS protein engineered for high
durability. By establishing a disulfide bond of L157C-G233C, full-activity
shelf life of E. coli MutS (eMutS) was prolonged
from 7 to 49 days and was further extended to 63 days via cellulose-bound
4 °C storage. In synthesis of 10 Cas9 homologues in-solution
and 10 xylose reductase (XR) homologues on-chip, iMICC reduced error
frequency to 0.64/Kb and 0.41/Kb, respectively, with 72.1% and 86.4%
of assembled fragments being error-free. By elevating base accuracy
by 37.6-fold while avoiding repetitive preparation of fresh enzymes,
iMICC is more efficient and robust than the wild-type eMutS, and it
is 6.6-fold more accurate and 26.7-fold cheaper than CorrectASE. These
advantages promise its broad applications in industrial DNA synthesis.