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Cells must detect and respond to molecular events such as the presence or absence of specific small molecules. To accomplish this, cells have evolved methods to measure the presence and concentration of these small molecules in their environment and enact changes in gene expression or behavior. However, cells dont usually change their DNA in response to such outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into a genomic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Therefore, through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered plasmids, which correspond to two orthogonal small molecule inducers that can be recorded with this system. We identified the optimal location of guide RNA binding to the Bxb1 attP integrase attachment site, and characterized the detection limits of the system by measuring the minimal small molecule concentration and shortest induction time necessary to produce measurable differences in array composition as read out by Oxford Nanopore long read sequencing technology.
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BACKGROUND:Previous studies have demonstrated that tissue Doppler echocardiography and two-dimensional speckle tracking echocardiography are minimally invasive imaging methods used to screen for coronary atherosclerotic heart disease.However,they are not highly sensitive and specific for patients with suspected heart disease presenting with normal ventricular wall motion or for patients with early coronary heart disease.The newly emerging three-dimensional longitudinal strain imaging technology can overcome these shortcomings and has become a relatively mature technique for quantitative assessment of myocardial function.OBJECTIVE:To investigate the early diagnosis value of left ventricular global longitudinal peak strain (LVGLPS)measured by three-dimensional longitudinal strain imaging technology for high-risk coronary atherosclerotic heart disease.METHODS:This is a single-center,open-label,diagnostic trial.Three hundred elderly patients suspected of coronary atherosclerotic heart disease receiving treatment at the Department of Ultrasound Medicine,Taihe Hospital of China from January 2013 to January 2018 are included in this study.These patients will be divided into three groups:low-risk group (n=100;≥ 70％ diameter stenosis in one or two branches of the right main coronary artery and the left circumflex artery),high-risk group (n=100;≥ 50％ diameter stenosis in the left main coronary artery or ≥ 70％ diameter stenosis in the left anterior descending branch),and control group (n=100;＜50％ diameter stenosis in the main coronary arteries and all branches).All patients will undergo conventional echocardiography followed by three-dimensional longitudinal strain imaging to measure the LVGLPS.The LVGLPS will be compared among the three groups.The primary outcome measure is the sensitivity of the LVGLPS for prediction of coronary atherosclerotic heart disease.The secondary outcome measures are:the specificity,positive predictive value,negative predictive value,positive likelihood ratio,negative likelihood ratio,and accuracy rate of the LVGLPS for prediction of coronary atherosclerotic heart disease;change in the LVGLPS;change in conventional echocardiography parameters;and change in the receiver operating characteristic curve for prediction of high-risk coronary atherosclerotic heart disease using the LVGLPS.This study will be performed in accordance with the Declaration of Helsinki.All patients have been informed of the study protocol and procedure and have provided written informed consent.This trial was approved by Taihe Hospital (Affiliated Hospital of Hubei University of Medicine) with the approval No.2013(03) in January 2013.Participant recruitment and data collection began in January 2013 and will continue through December 2017.Outcome measure analysis will be performed and the trial will be completed in January 2018.Results will be disseminated through presentations at scientific meetings and publications in peer-reviewed journals.This trial was registered with ClinicalTrial.gov (identifier:ChiCTR-DDD-17012839).DISCUSSION:The findings from this study will help to confirm that three-dimensional longitudinal strain imaging technology is highly sensitive and specific for patients with abnormal coronary arteries with suspected coronary heart disease but who present with normal ventricular wall motion.The change in the LVGLPS contributes to early diagnosis of high-risk coronary atherosclerotic heart disease in elderly patients.This helps clinicians to diagnose early coronary heart disease and take timely strategies to avoid serious cardiovascular events as much as possible.