Method for human PD1 gene specific knockout through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat) and sgRNA(single guide RNA)for specially targeting PD1 gene
A pgl3-u6-hpd1sg, specific technology, applied in the field of genetic engineering, can solve problems such as laborious, time-consuming and expensive antibody drugs
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[0060] Example 1 Design and synthesis of sgRNA for specifically targeting PD1 gene in CRISPR-Cas9 knockout human PD1 gene
[0061] 1. Design of sgRNA targeting human PD1 gene:
[0062] (1) Select the 5'-GGN(19)GG sequence on the PD1 gene, if there is no 5'-GGN(19)GG sequence, 5'-GN(20)GG or 5'-N(21)GG also can.
[0063] (2) The targeted site of sgRNA on the PD1 gene is located in the exon of the gene, which is more likely to cause fragment deletion or frame-shift mutation, so as to achieve the goal of complete gene inactivation.
[0064] (3) The target site of sgRNA on the PD1 gene is located on the common exons of different splicing forms.
[0065] (4) Use BLAT in the UCSC database or BLAST in the NCBI database to determine whether the target sequence of sgRNA is unique and reduce potential off-target sites.
[0066] According to the above method, we designed a total of 180 sgRNAs targeting the human PD1 gene. The sequences are as shown in SEQ ID NO.24-203 in the sequence table.
[0067...
Example Embodiment
[0087] Example 2 Using CRISPR-Cas9 to specifically knock out the human PD1 gene (the sgRNA used to target the PD1 gene is shown in Sequence Listing 52)
[0088] 1. The linearized sequence is the pGL3-U6-sgRNA plasmid shown in SEQ ID NO.10 in the sequence table.
[0089] The digestion system and conditions are as follows:
[0090] 2μg pGL3-U6-sgRNA (400ng / μl);
[0091] 1μl CutSmart Buffer;
[0092] 1μl BsaI (NEB, R0535L);
[0093] Replenish water to 50μl, incubate at 37°C for 3 to 4 hours, shake and centrifuge at intervals to prevent droplets from evaporating to the tube cap.
[0094] After the digestion is completed, use AxyPrep PCR Clean up Kit (AP-PCR-250) to purify and recover it into 20-40μl sterile water.
[0095] 2. After denaturation and annealing, the double-stranded sgRNA oligonucleotides (the Forward oligo and Reverse oligo sequences of which are shown in SEQ ID NO. 1 and 2 in the sequence table respectively) that can be connected to the U6 eukaryotic expression vector are combin...
Example Embodiment
[0125] Example 3 Using CRISPR-Cas9 to specifically knock out the human PD1 gene (the sgRNA used to target the PD1 gene is shown in SEQ ID NO.82 in the sequence list)
[0126] 1. The linearized sequence is the pGL3-U6-sgRNA plasmid shown in SEQ ID NO.10 in the sequence table.
[0127] The digestion system and conditions are as follows:
[0128] 2μg pGL3-U6-sgRNA (400ng / μl);
[0129] 1μl CutSmart Buffer;
[0130] 1μl BsaI (NEB, R0535L);
[0131] Replenish water to 50μl, incubate at 37°C for 3 to 4 hours, shake and centrifuge at intervals to prevent droplets from evaporating to the tube cap.
[0132] After the digestion is completed, use AxyPrep PCR Clean up Kit (AP-PCR-250) to purify and recover it into 20-40μl sterile water.
[0133] 2. After denaturation and annealing, the double-stranded sgRNA oligonucleotides (the forward oligo and reverse oligo sequences of which are shown in SEQ ID NO. 3 and 4 in the sequence table) that can be connected to the U6 eukaryotic expression vector are combi...
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