A gRNA targeting HPK1 and a method for editing HPK1 gene
一种HPK-1、基因的技术，应用在分子生物学和免疫学领域，能够解决肿瘤杀伤力不足、T细胞衰竭、活性降低等问题
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Embodiment 1
[0321] Design and synthesis of embodiment 1.gRNA
[0322] 1. Design of guide RNA
[0323] The plasmid loaded with gRNA is pUC57kan-T7-gRNA, the gRNA targeting HPK1 was designed, and the gRNA targeting PD1 was used as a control. The sequence of the gRNA specifically targeting HPK1 paired with the genome is GACCTGGTGGCACTGAAGA (located at the second exon of HPK1, SEQ ID NO: 1); the sequence of the gRNA specifically targeting PD1 paired with the genome is GGCCAGGATGGTTCTTAGGT (located at the second exon of PD1 The first exon of , SEQ ID NO: 2).
[0324] HPK1 gRNA: F: 5′-TAGG GACCTGGTGGCACTGAAGA-3′ (SEQ ID NO: 3)
[0325] R: 5'-AAAC TCTTCAGTGCCACCAGGTC-3' (SEQ ID NO: 4)
[0326] PD1 gRNA: F: 5′-TAGG GGCCAGGATGGTTCTTAGGT-3′ (SEQ ID NO: 5)
[0327] R: 5'-AAAC ACCTAAGAACCATCCTGGCC-3' (SEQ ID NO: 6).
[0328] Synthesize the gRNA coding strand and the complementary strand by annealing the two DNA strands of HPK1 gRNA and PD1 gRNA to form a double-stranded DNA template, which is in...
Embodiment 2
[0334] Recombinant expression and purification of embodiment 2.Cas9 protein
[0335] Humanized Cas9 (sequences are provided in SEQ ID NOS: 18 and 19) was prepared according to methods reported in the literature. Chang, N et al., Cell Res. 23:465-472 (2013), the contents of which are incorporated herein by reference in their entirety, including the sequence of the humanized, codon-optimized Cas9 cDNA and the protein sequence reported therein. Briefly, the plasmid used to load Cas9 was PGEX4T-1, and a codon-optimized full-length human cas9 cDNA was obtained by PCR. The template is the plasmid pUC19-T7-CAS9. A nuclear localization signal (NLS) was added to the 5' and 3' ends of the Cas9 sequence to facilitate nuclear import of the cas9 protein.
[0336] After successfully constructing the plasmid, express the CAS9 protein by Escherichia coli, purify it through a GST column, concentrate and collect the protein, cut the GST tag by thrombin, and observe a protein band near the 160...
Embodiment 3
[0337] Example 3. In vitro expansion and transfection of human peripheral blood mononuclear cells
[0338] use Sodium diatrizoate solution was used to extract human peripheral blood mononuclear cells by centrifugation and rapid separation. CD3-positive peripheral blood mononuclear cells were sorted using CD3 magnetic beads, T cells were activated using CD3 / 28 antibody, and incubated in LONZA-X-VIVO 15 medium containing 100U / ml IL-2 at 37℃ / 5% CO 2 Viral transfection and electroporation were performed after 2 days of culture under the conditions of .
[0339] Mix cas9 protein and gRNA mRNA in a certain ratio, and let stand at room temperature for 10 minutes, meanwhile, mix 4.0×10 6 CD3+ T cells (activated with CD3 / CD28 antibody for 48 hours in vitro) were transferred to a 15ml centrifuge tube. Cells were centrifuged at 500 x g for 5 minutes and then resuspended in 400 μl optimized medium (mixture containing cas9 and gRNA). The cell mixture was transferred to a BTX cuvette ...
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