Construction of Genetic Sense-and-Respond Modules to Detect Viral Infection

Project Overview

Genetic diseases such as sickle cell disease, muscular dystrophy, Huntington's disease, and cancer are caused by a mutation or handful of mutations in the DNA of a cell. These diseases could be cured if the incorrect DNA bases were changed to the correct bases. Until recently, making these genetic alterations to correct a cell's DNA was a nearly impossible task. However, the introduction of clustered regularly interspaced short palindromic repeats (CRISPR) as a targeted, programmable gene editing technique made this goal attainable. CRISPR-based treatments for genetic diseases are currently under development, with some reaching the stage of clinical trials in humans. While these developments are promising as future genetic disease treatments, it is vital to understand the side effects that a cell experiences as a result of CRISPR gene editing. Some reports show that CRISPR gene editing leads to deletions of large segments of DNA; others show that CRISPR creates unintended edits in DNA. Here, we used a suite of genetic and molecular biology techniques to characterize the stress responses that cells activate when CRISPR components are present. Cellular tolerance of CRISPR activity depends on what type of cell it is, the amount of CRISPR-associated (Cas) protein expressed in the cell, and the duration of Cas exposure. Transformed cells, such as HEK-293 human embryonic kidney cells, activate pathways related to oxidative stress, cell cycle arrest, and DNA repair upon Cas9 expression. Non-transformed MCF10A human mammary epithelial cells on the other hand do not activate the same stress response pathways when Cas9 is expressed but readily undergo senescence or apoptosis. These results pave the way for a more robust understanding of how CRISPR gene editing affects individual cells, and this understanding will inform our development and use of CRISPR-based therapeutics to avoid unanticipated negative effects of genetic engineering.

Mission Impact

CRISPR has revolutionized the life sciences, enabling precise edits, insertions, and deletions to almost any sequence of DNA. The opportunities to use this technology in therapeutics that cure genetic diseases are vast. It is also important to understand the limitations of this technology and how genetic engineering techniques like CRISPR affect cell health. These limitations inform therapeutic development as well as national security issues. This project has brought additional expertise in advanced genetic engineering methods, especially CRISPR/Cas engineering, to Lawrence Livermore National Laboratory and has given us experience with these methods so that we have a clearer understanding of their utility in various applications. This work also furthers Livermore's biomarker identification and characterization work by using proteomics and transcriptomics to identify makers of cellular stress in response to CRISPR gene editing activity. These biomarkers can be used in future programs that evaluate gene editing efficacy and move the field toward predicting the outcome of gene editing therapeutics based on the target DNA.

Publications, Presentations, and Patents

Collette, N.M., et al. 2019a. "Forensic Analysis of the Metabolic Cost of Cas9." Genome Engineering: From Mechanisms to Therapies, Keystone Conference, Victoria, BC, Canada, 19-23 February 2019. LLNL-POST-767839.

2019b. "Forensic Analysis of Cas9 Reveals Cellular Stress." Genome Engineering: Frontiers of CRISPR/Cas, Cold Spring Harbor Laboratory Meeting, Cold Spring Harbor, NY, 10-13 October 2019. LLNL-POST-793104.