grna oligonucleotides  (New England Biolabs)

Journal: bioRxiv

Article Title: DNA Damage Response Proteins Are Involved in the Formation of Defective HIV-1 Proviruses

doi: 10.64898/2026.03.31.715508

Figure Lengend Snippet: A) Flow cytometry gating strategy for K562 VPR cells transduced with CRISPRa-gRNA and LTatC[M]L lentiviral vectors. Example shown for CRISPRa-HLTF technical replicates. B) Single mCherry+/double-positive Cerulean+/mCherry+ (mCherry/Dp) cell ratio for candidate genes in K562-VPR cells transduced with corresponding CRISPRa-gRNA and LTatC[M]L. Data shown relative to non-targeting control gRNA, marked by the dotted line. Each dot represents one technical replicate. C) mCherry/Dp ratio for candidate genes in K562 cells transfected with corresponding siRNA and transduced with LTatC[M]L. From the left: transfection with siRNAs targeting single genes; transfection with combination of siRNAs targeting two genes. Data shown relative to scrambled control siRNA, marked by the dotted line. Each dot represents one technical replicate. All data was statistically tested using mixed-effect logistic regression model, p value <0.1 was considered significant. **** = p <0.001, *** p <0.01, ** p <0.05.

Article Snippet: The Golden Gate reaction contained 25 ng backbone plasmid, 1 μl of 1:10 diluted annealed gRNA oligonucleotides, 0.5 μl BsmBI-v2, 0.5 μl T4 DNA ligase (Thermo Scientific, EL0011), 1 μl 10 mM ATP (New England Biolabs, P0756S), 1 μl NEB Buffer r3.1 (New England Biolabs), and nuclease-free water to the final reaction volume.

Techniques: Flow Cytometry, Transduction, Control, Transfection

Journal: Nature

Article Title: Engineered prime editors with minimal genomic errors

doi: 10.1038/s41586-025-09537-3

Figure Lengend Snippet: a , Assay schematic, where paired gRNAs induce DNA breaks that lead to perfect junctions for nuclease variants that cut at the canonical position between the +1 and −1 nucleotides. Shifts in the cut position lead to retention of additional sequences within the junction, and the frequencies of these retained sequences are used to determine the nick shift frequencies for each nuclease variant. b , Design of gRNA pairs and expected sequences of the junction products at the CXCR4 and EMX1 loci, with the expected retained sequences due to shifts in nick position indicated. c , Rates of the top genomic sequences resulting from editing with different Cas9 variants at the junction for the CXCR4 locus. Only reads with the reference sequence or retained sequences (insertions boxed in red) are depicted. The most frequent allelic sequences above 0.05% of reads are displayed along with sequencing reads and percentages out of all reads. d , DNA nick position frequencies for nuclease variants averaged at the CXCR4 and EMX1 loci. The relative frequencies of retained sequences in the junction products are used to estimate the frequencies of nick shifts of corresponding length. Data were analysed by deep sequencing and represent a single replicate (in c ) or means of n = 6 independent replicates (in d ).

Article Snippet: The nicking gRNA spacer sequence oligos, listed in Supplementary Table , were phosphorylated with T4 polynucleotide kinase (NEB) and cloned into gRNA cloning backbone by Golden Gate cloning with BpiI digestion.

Techniques: Variant Assay, Genomic Sequencing, Sequencing

Journal: Nature

Article Title: Engineered prime editors with minimal genomic errors

doi: 10.1038/s41586-025-09537-3

Figure Lengend Snippet: a , Assay schematic for end degradation comparing nuclease variants, where paired gRNAs induce DNA breaks that lead to perfect junctions for non-degraded DNA ends and deletions in the junction products indicate degradation of the DNA ends. The length of each deletion to the non-PAM-side or PAM-side of the junction indicates the extent of degradation, and the frequencies of these deletions indicate the frequency of degradation. b , Design of gRNA pairs and expected sequences of the junction products at the EMX1 and CXCR4 loci, with the direction of degradation-associated deletions indicated. c , Rates of the top genomic sequences resulting from editing with different Cas9 variants at the junction for the EMX1 locus. Only reads with the reference sequence or deletions are depicted. The most frequent allelic sequences above 0.05% of reads are displayed along with sequencing reads and percentages out of all reads. d , Assay schematic for end degradation comparing prime editor variants, where a paired pegRNA and ngRNA induce DNA nicks that lead to large deletions between homologies in the flaps flanking the nicks for non-degraded ends but cannot produce deletions if the nicked end flaps are degraded. The pegRNA can also install a larger edit that is used as an activity marker for the prime editor, and degradation is quantitated as the ratio of the activity marker edit to the flap homology deletion. e , Design of the pegRNA and ngRNA pair at the AAVS1 locus, with the extensive GC-rich homologies indicated. f , Deletion frequencies by position within the targeted locus comparing several prime editor variants, indicating dominant deletions between homologous sequences flanking the nicks. Data were analysed by deep sequencing and represent a single replicate (in c ) or means of n = 3 independent replicates (in f ).

Article Snippet: The nicking gRNA spacer sequence oligos, listed in Supplementary Table , were phosphorylated with T4 polynucleotide kinase (NEB) and cloned into gRNA cloning backbone by Golden Gate cloning with BpiI digestion.

Techniques: Genomic Sequencing, Sequencing, Activity Assay, Marker