Method for detecting off-target effect of CRISPR-Cas9
An off-target effect and off-target technology, applied in the field of genetic engineering, can solve the problems of low sensitivity, insufficient accuracy, and inability to guarantee the labeling of break sites, and achieve the effect of improving accuracy and sensitivity and improving safety.
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Embodiment 1
[0031] Human PD1 gene CRISPR / Cas9 gene editing sgRNA1 off-target site detection:
[0032] Step 1: Prediction of off-target sites and synthesis of PCR amplification primers. The potential off-target sites of the PD1 gene editing sgRNA1 used were predicted by the software seqmap. Specifically, the off-target site of sgRNA1 is predicted by software, and the prediction software (website) includes seqmap, CRISPOR, CRISPR finder, CRISPR Design, sgRNAcas9, CRISPRdirect, COSMID, Off-Spotter, E-CRISP, etc., but not limited thereto. The 6 potential off-target sites with the highest score (highest possibility of off-target) were screened, and the information of the off-target sites is shown in Table 1. And derive PCR amplification primers (table 2) for these off-target sites;
[0033] Table 1 Screening of human PD1 gene CRISPR / Cas9 gene editing sgRNA1 off-target sites
[0034]
[0035] Underlined bases in Table 1 indicate mismatches
[0036] Table 2 Off-target site PCR primer info...
Embodiment 2
[0059] Human PD1 gene CRISPR / Cas9 gene editing sgRNA2 off-target site detection:
[0060] Except that Table 1 is replaced by Table 3 in Comparative Example 1, and Table 2 is replaced by Table 4, all the other steps are the same.
[0061] Table 3 Screening of human PD1 gene CRISPR / Cas9 gene editing sgRNA2 off-target sites
[0062]
[0063] Table 3 Underlined bases indicate mismatches
[0064] Table 4 Off-target site PCR primer information
[0065]
[0066] The results showed that, except for single base mutations found in OT7 and OT13, there were no insertions, deletions and point mutations in other potential off-target sites. Sequence comparison found that both the unedited sample and the edited sample had the same point mutation, and the mutation site was not in the potential off-target region, such as figure 1 with 2 As shown, thus indicating that the sgRNA does not have off-target effects.
Embodiment 3
[0068] Human PD1 gene CRISPR / Cas9 gene editing sgRNA3 off-target site detection:
[0069] Except that in step 1 of Comparative Example 1, the use of seqmap to predict off-target sites was replaced by CRISPOR to predict off-target sites, Table 1 was replaced with Table 5, and Table 2 was replaced with Table 6, the rest of the steps were the same.
[0070] Table 5 Screening of human PD1 gene CRISPR / Cas9 gene editing sgRNA3 off-target sites
[0071]
[0072]
[0073] Table 5 Underlined bases indicate mismatches
[0074] Table 6 Off-target site PCR primer information
[0075]
[0076] The results showed that in OT16, a deletion of 3 bases was found in all three edited samples (such as image 3 shown).
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