Genetic Engineering Publications - GEG Tech top picks

In a recent study published in Proceedings of the National Academy of Sciences, researchers developed a new assay for the detection of SARS-CoV-2. For the test, CRISPR systems were coupled to loop-mediated isothermal amplification (LAMP) or recombinase polymerase (RPA) amplification to boost sensitivity and improve detection at attomolar levels. In addition, reverse transcription (RT)-RPA/LAMP systems coupled with Cas13 systems have been developed for the detection of SARS-CoV-2. RT-LAMP has become the preferred method due to its high sensitivity, low cost and ease of use. Since these systems use functional Cas enzymes at 37°C and LAMP requires high temperatures (55°C to 65°C), the RT-LAMP/RPA assays coupled with CRISPR. The multiplexed assay therefore has a thermophilic Cas13a isolated from Thermoclostridium caenicola (TccCas13a) and uses it for the detection of SARS-CoV-2 using fluorescent amidite-labelled RNA and has developed an AapCas12b-based RNase P detection using HEX-labelled ssDNA reporters. The researchers achieved simultaneous detection of RNase P and SARS-CoV-2 without any interference from the fluorescence signals. A mobile phone application was developed to collect and interpret the fluorescence viewer readings at low cost. 

In this work the scientists employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5′ and 3′ LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells.

A group of researchers set out to characterize the genotypic abnormalities induced by Cas9 in human cells. To do this, they studied the tRNA gene. These researchers deleted two tRNA genes from the genomes of hyperploid human hepatocellular carcinoma (HepG2) and haploid chronic myeloid leukemia (HAP1) cells using the CRISPR/Cas9 system with dual gRNAs. The underlying alterations that caused rearrangement of the CRISPR/Cas9-targeted region were assessed by a customized de novo sequence assembly approach. Although a genomic region of interest was cleaved by Cas9 in this study, the cleaved fragment was duplicated, inverted, and inserted locally into the genomes of both HepG2 and HAP1 cells. The study also demonstrated the successful integration of exogenous DNA fragments into HepG2 cells.

Furthermore, the aberrant target-derived DNA fragments were shown to be still functional, tagged with active histones, and bound by RNA polymerase III. This highlights the fact that CRISPR/Cas9-based genomic engineering can lead to adverse effects on the target, and that inversion and duplication events can occur at the same time. In conclusion, this study reveals the complex genomic alterations that accompany CRISPR/Cas9 deletions.

Here the author demonstrate specific and orthogonal two-color labeling of repetitive sequences in living human cells using this method. By attaching the MS2 or PP7 aptamers to different locations on the sgRNA, they found that extending the tetraloop and stem loop 2 of the sgRNA with MS2 or PP7 aptamers enhances the signal-to-background ratio of chromatin imaging.

In this work, the authors adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Their results demontrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.