The genomic DNA was extracted by  TIANamp Genomic DNA Kit (DP304, Tiangen). all DNA was used toPCR for enrichment of sgRNA. The sequences of primers for sgRNA amplification were as follows: forward, 5'-AGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAG-3'; reverse, 5'-AAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCT-3'. Firstly， the PCR products'  terminal were repaired and the 3'  A tail were added to the end, the DNA template was connected to the splice at both ends of the fragment, to prepare PCR-free library The Human CRISPR Knockout Pooled Library sequnence