...Wyatt and Dale Ramsden, UNC at Chapel Hill. One advantage to using the CRISPR/Cas system for genome engineering...Persistence of a Chromosome Break Diminishes Its Mutagenic Potential. PLoS Genet 5:e1000683 . https://doi.org...End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis. mBio 11: . https://doi.org/10.1128...Genome-Wide Control for Large-Scale Functional Screens        ...

... in yeast Base editing CRISPRi in Caulobacter crescentus Multiplexed gRNA for human pluripotent stem cells...that controls repression of gene expression in Saccharomyces cerevisiae. The system consists of dCas9, the...editing. Using several rapid and cost-effective screening methods, David Liu’s lab identified and validated... base editor plasmids CRISPRi in Caulobacter crescentus Figure 1: Serial dilutions of CRISPRi in...adapted from Guzzo et al., 2020. Caulobacter crescentus is a popular model organism for studying the ...ctrA sgRNA and saw that the levels of ctrA mRNA decreased after 20 minutes in xylose (which controls expression... 2: The components of Casilio-ME1. Image from Taghbalout et al., 2019. Fusing epigenetic modifiers...

...Figure 2: CRISPR R-loop. Created with BioRender.com. Base editors take advantage of this single-stranded...nucleobase component of the nucleoside inosine. Created with BioRender.com. Although it was a huge ...have scientists solved this problem? By taking advantage of repair pathways within the cell — in some cases...it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate strands called an R-loop (...activity window restricted to the stretch of ssDNA created by Cas9 binding. Pro tip! In CRISPR, the non-target...BER process was excising the incorrect U base, creating an “abasic,” or empty, DNA site, and then replacing...in the same journal on the same day.) This team created a similar construct that they called a Glycosylase...

...the human gut bacteria B. thetaiotaomicron, Mycobacterium, Pseudomonas, and E. coli. However, many non-model...for CRISPRi transfer Mobile-CRISPRi offers two advantages over existing bacterial CRISPRi methods. First...better suited for genome-wide CRISPRi library screening experiments where maintaining library diversity...of antibiotics are often essential genes which creates a challenge when studying them since their complete...

...DNA end of the break is resected by nucleases to create a ssDNA 3’ overhang. This will serve as both a ...considerations for designing a repair template to create mutations HDR donors can contain insertable sequences...circular plasmid templates is generally low; to increase the frequency of edits, researchers have designed...complementary ssDNA. Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25... chromosomal aberrations. Pfeiffer P, et al. Mutagenesis (2000). PubMed. Multiple Pathways of Recombination...Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae. Paques F and Haber JE. Microbiology...Frequency of Homology-directed Repair (HDR) for Increased Efficiency of CRISPR/Cas-mediated Transgenesis...

...majority of plasmids contain either an ampicillin or kanamycin resistance gene. Every step along the way involves...and prepare fresh media with antibiotics at each stage. The barcodes and LIMS help us track each sample...deposit. Using barcoded tubes is part of Addgene’s secret to success. It helps us quickly process samples...

...promoters and create sophisticated patterns of UAS-driven gene expression. To further increase the complexity...system into a repressible system, Gossen and Bujard created the tetracycline-controlled transactivator (tTA...bacterial operons. A chimeric transactivator, cTA, was created by fusing CymR to the activation domain VP16. cTA...GAL4 gene downstream of a tissue- or developmental stage-specific promoter and design a UAS reporter construct...state, and co-expression of the inducer QF can increase QUAS driven expression by ~3,300-fold in Drosophila...genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197...