Journal: Science Advances

Article Title: Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing

doi: 10.1126/sciadv.abo7676

Figure Lengend Snippet: The genotype of each colony was identified by S-R NGS, L-R PCR, and ddPCR to account for the dropout of LD alleles. ( A ) S-R and L-R PCR primer designs to amplify the region around the R-66S cut site on HBB . ( B ) Genotype results based on S-R NGS with the combination of the three assays for 100 clones derived from S-HUDEP2 treated with R-66S RNP. ( C ) Genotype results based on S-R NGS with the combination of the three assays for 72.5 ± 12.0 erythroid colonies derived from SCD HSPCs treated with R-66S RNP. The use of S-R NGS significantly overestimated the percentage of small INDEL (SI) alleles compared with that identified using the combination of three assays. 23.4 ± 0% LD alleles occurred in 40.1 ± 0.8% colonies, which caused a significant reduction of HBB copy numbers in RNP-treated SCD HSPCs. ( D ) Genotype results for 79 ± 7 erythroid colonies derived from SCD HSPCs treated with both R-66S RNP and the corrective ssODN donor. A total of 11.8 ± 0.8% of alleles had LD in 18.5 ± 2.9% of colonies. S-R NGS overestimated the percentage of homozygous HDR colonies (35.4 ± 11.3%) compared to that obtained by the combination of three assays (27.2 ± 7.5%).

Article Snippet: To identify clones carrying a LD, the 5.44-kb region containing the on-target cut site at the center was amplified using L-R PCR [LongAmp Hot Start Taq DNA Polymerase; New England Biolabs (NEB), M0534S].

Techniques: Clone Assay, Derivative Assay

Journal: Science Advances

Article Title: Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing

doi: 10.1126/sciadv.abo7676

Figure Lengend Snippet: ( A ) Schematics of the LongAmp-seq assay. The LongAmp-seq assay is based on L-R PCR amplification around the Cas9 cut site followed by tagmentation, adaptor extension, and Illumina paired-end deep sequencing. A bioinformatic pipeline was developed for sequence merging, alignment, filtering, and identification of repair outcomes. ( B ) The read coverage pattern of the R-66S RNP-treated SCD HSPCs, normalized by that of the control sample. LongAmp-seq (red) gave similar normalized depletion patterns surrounding the R-66S on-target cut site compared with that obtained by SMRT-seq (blue) ( C ) Small INDEL profile plot from R-66S RNP-treated samples, showing the overlap between SMRT-seq and LongAmp-seq results. ( D ) High correlation between the percentage of small INDEL in unsplit reads quantified by SMRT-seq and LongAmp-seq. ( E ) LongAmp-seq–identified LD patterns were mapped relative to the Cas9 cut site. Representative LongAmp-seq LD profile from R-66S RNP-treated SCD HSPCs from Donor #2. ( F ) High correlation between the percentage of LD quantified by SMRT-seq with UMI and the percentage of LD reads measured by LongAmp-seq. In (D) and (F), biological replicates for each sgRNA were indicated by symbols. n = 2 for R-66S RNP, R-66S RNP + ssODN, SD-02 RNP, and BCL11A RNP and n = 1 for R-02 RNP. ( G ) The LD patterns identified by SMRT-seq and LongAmp-seq for the R-66S RNP sample were plotted on the basis of the location of midpoint of LDs ( x axis) and LD sizes ( y axis). The LDs identified by LongAmp-seq had a high level of overlap (96%) with that by SMRT-seq.

Article Snippet: To identify clones carrying a LD, the 5.44-kb region containing the on-target cut site at the center was amplified using L-R PCR [LongAmp Hot Start Taq DNA Polymerase; New England Biolabs (NEB), M0534S].

Techniques: Amplification, Sequencing