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Fig. 1 | BMC Genomics

Fig. 1

From: CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening

Fig. 1

Overview of CORALINA (comprehensive gRNA library generation through controlled nuclease activity). a Three CORALINA libraries (L1, L2, L3) from genomic DNA of human and mouse each were generated and analyzed by NGS to compare conditions and corroborate the reproducibility of the method. b In principle, any type of double stranded DNA can be used as a source for CORALINA library generation. The DNA is fragmented through controlled digestion with MNase and ligated to linker oligos containing only one blunt end, but no phosphate residue. Consequently, linkers aligning in wrong orientation or to linker-oligos instead of fragments are excluded. PCR amplification allows size selection of those sequences containing two different linker sequences. Three different linker sequences (L1, L2, L3) have been used for bulk incorporation into gRNA-PLKO.1 using Gibson Assembly (p: phosphate residue; blue, grey lines: linker sequences and homology regions on gRNA-PLKO.1; orange lines: protospacer sequences, small arrows: primer sequences). c The analyzed representative samples of the three generated human CORALINA libraries have little sequence overlap indicating large library complexity. Shown in numbers are the unique occurrences of protospacers longer than 17 bps

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