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. Author manuscript; available in PMC: 2019 Jun 4.
Published in final edited form as: Nat Protoc. 2018 Jan 11;13(1):307–323. doi: 10.1038/nprot.2017.131

Figure 4. Cloning a CRISPR library into pU6-DHFR.

Figure 4.

(a) Oligonucleotide arrays contain sgRNAs (highlighted in blue) flanked by regions homologous to pU6-DHFR (green and orange) and sequences for the isolation of sgRNAs targeting subsets of genes (gray). (b) sgRNAs plus regions bearing homology to pU6-SAG1 are amplified using Primer 5 and Primer 6 (Table 1). The resulting DNA fragments are inserted into BsaI-linearized pU6-DHFR using Gibson Assembly. See the Supplementary Methods for a detailed procedure.