The authors demonstrate that sample cross talk due to combinatorial indexes can be corrected by using unique adapters containing unique molecular identifiers (UMIs). Their results suggest that such adapters are critical to reducing false positive rates in sensitive applications, such as somatic variant discovery. The adapter design presented in this article uses dual matched indexes. xGen Dual Index UMI Adapters—Tech Access incorporate an updated design, using dual unique indexes based on Illumina’s best practice guidelines.
Here, researchers deliver Cas9 ribonucleoprotein (RNP) to various cell types, including human primary CD4+ T cells, via a novel microfluidic cell deformation-based method.
The authors use DNA origami with GFP antibodies to provide a system for calibrating fluorophore and antibody labeling efficiency. The method should allow researchers to quantify protein copy number within cells using super-resolution microscopy.
Li MA, Amaral PP, Cheung P, Bergmann JH, Kinoshita M, Kalkan T, Ralser M, Robson S, von Meyenn F, Paramor M, Yang F, Chen C, Nichols J, Spector DL, Kouzarides T, He L, Smith A.
A lncRNA fine tunes the dynamics of a cell state transition involving Lin28, let-7 and de novo DNA methylation
This study shows that in vitro-assembled, dual Alt-R Cas9 RNPs coupled with microhomology repair templates enable efficient gene manipulation in different genetic backgrounds of A. fumigatus.
This publication reports a screening effort to uncover novel regulators of vertebrate spindle orientation. The authors describe a method for efficient gene knockout in chick embryos which employs in ovo electroporation of Alt-R CRISPR-Cas9 components (crRNA and tracrRNA).
xGen Lockdown Probes and Blocking Oligos were used to generate a custom gene panel that targeted 382 cancer-relevant genes in multiple cancer types. The panel provided target enrichment across 605 circulating tumor (ctDNA) samples.
Exome sequencing with the xGen Exome Research Panel established a molecular genetic diagnosis of TARP syndrome for a neonatal patient in whom traditional testing methods were uninformative. Sequencing of the proband, mother, and father showed a previously unreported, maternally-inherited alteration in an RNA-binding motif protein.This allowed efficient diagnosis and future reproductive options for the parents.
26 paired samples (FFPE-derived tumor samples + frozen, PMBC-derived normal samples) were sequenced by exome hybrid capture using the xGen Exome Research Panel. RNA sequencing was also performed using the panel. The study demonstrates that improvements to the efficacy of immune-mobilizing therapies require the genetic profiling of both the tumor and immune system.
Researchers from the University of Washington and TwinStrand Biosciences describe a targeted sequencing approach called CRISPR-DS, which couples a previously described method known as duplex sequencing with CRISPR-Cas9 system for target selection. Alt-R CRISPR-Cas9 RNAs were used in in vitro digestion to fragment input genomic DNA at specified locations, followed by size selection. Compared to standard duplex sequencing approach, CRISPR-DS resulted in 20-fold improvement of on-target rate using only minimal amounts of input DNA.
Combining CRISPR-Cas9 genome editing and HiBiT reporter technologies, this study describes an approach to efficiently tag endogenous proteins with a small luminescent peptide. The researchers achieved rapid, high integration efficiency and assay sensitivity via electroporation of a pre-assembled Alt-R Cas9 RNP complex and ssODN templates, ordered as IDT Ultramers. This enabled quantification of protein levels in the mixed population of edited cells without requiring clonal isolation.
Choi YJ, Lin CP, Risso D, Chen S, Kim TA, Tan MH, Li JB, Wu Y, Chen C, Xuan Z, Macfarlan T, Peng W, Lloyd KC, Kim SY, Speed TP, He L.
Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells
This study provides an example of disrupting endogenous gene expression in mouse MC38 cells via electroporation of a pre-assembled Alt-R Cas9 RNP complex. By generating a tumor cell line in which both alleles of transmembrane protein CD47 are knocked out, the researchers show that increased sensing of tumor-derived DNA (achieved by CD47 blockade) primarily occurs in dendritic cells but not in microphages. These findings shed light on the molecular mechanism underlying immune invasion of tumor cells.
Researchers from Virginia Commonwealth University describe a novel CRISPR-Cas9 mediated “nanomapping” approach, which may fill technical gaps that are poorly addressed by existing DNA-mapping techniques. Using Alt-R CRISPR guide RNAs and high-speed atomic force microscopy (HS-AFM), they report successful detection and precise mapping of BCL2-IGH translocations in clinical samples derived from follicular lymphoma patients.
The authors of this paper describe Easi-CRISPR, a robust and efficient strategy for targeted DNA cassette insertion in mice. The international consortium of 7 research teams injected mouse zygotes with long single-stranded DNA donors (Megamer Single-Stranded DNA Fragments) and pre-assembled Cas9 ribonucleoprotein complexes (Alt-R crRNA, tracrRNA, and Cas9 nuclease), and obtained successful knock-in at 13 loci.
This publication details the process of designing knock-out and knock-in CRISPR experiments for the generation of new mouse mutants. It outlines proper preparation of Cas9 ribonucleoprotein, as well as procedures for delivering the complex to mouse zygotes by way of microinjection and electroporation.
This publication from the laboratory of Dr Eric Kmiec highlights the advantages of using CRISPR-Cas9 ribonucleoprotein for DNA cleavage along with single-stranded DNA oligonucleotides for repair of single base mutations, and examines the mechanism of repair in greater detail.
Researchers from the Doyon laboratory in Quebec describe a method for multiplexing CRISPR guide RNAs, which enables a coselection strategy that can be used to enrich populations of successfully-edited cells following non-homolgous end joining or homology-directed repair.