Recording of elapsed time and temporal information about biological events using Cas9

An Analysis of gRNA Sequence Dependent Cleavage Highlights the Importance of Genomic Context on CRISPR-Cas Activity

“…Both of these datasets utilize different sgRNA scaffolds that modify the four consecutive thymines present towards the 5’ end of the scaffold. 20,23 As four thymines in a row is a known transcription terminator for the eukaryotic RNA polymerase III, the result of this modification is higher expression of the gRNA. 28 This suggests that increased gRNA expression reduces the impact of context on activity.…”

“…We report activity as it was reported in the original dataset but have inverted the sign on several datasets to ensure that in our comparisons more positive numbers correlate with more active gRNA. Datasets for which we inverted the sign include Xu et al 2015 6 , Moreb et al 2017 22 , Schwartz et al 2019 11 , Moreb et al 2020 16 , and Park et al 2021 23 . The data in Supplementary Files 2-6 include this sign inversion.…”

“…Similarly for Kim et al 2020b 20 , we only took data from gRNA Library B and we excluded repeat gRNA. From Park et al 2021 23 , we only took data from Library 1. Schwartz et al 2019 11 performed library experiments in the presence and absence of the native NHEJ repair pathway.…”

CRISPR Clocks: The Times They Are a-Changin’

“…Since the initial completion of the Human Genome Project 20 years ago, there have been widespread efforts to systematically interrogate the functional characteristics of the human genome. 1 The boom in the field of functional genomics has, in part, been fueled by advances in the field of genome engineering using CRISPR-Cas9. [2][3][4][5][6][7][8][9][10] CRISPR-based gene-editing tools have contributed greatly to our understanding of complex genetics in model systems and in humans.…”

“…Park et al have creatively exploited a classical CRISPR system and adapted it to the concept of the molecular clock in the context of both in vitro and in vivo mammalian cell models. 1 The researchers present a method in Cell in which biological events can be temporally recorded into a DNA sequence with predictable kinetics according to an exponential decay model. Their proposed synthetic ''DNA Clock'' system opens the door to developing living cellular devices that record the timing and duration of events such as chemical or pharmacological exposure, disease progression, tissue development, or other cellular decision making.…”