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The Liu Library Data file includes the following supplemental files.
liu-library-drosophila-summary.docx - A short description summarizing the library and screening
count-seq-FASTQ-multiple-v2.pl - a perl script for use after the library has been amplified and submitted to next-generation sequencing. This script will take the split FASTQ files generated by FASTQ_splitter.pl, and a list of sgRNAs to compare (i.e. sgRNA_library.out), and count the instances of each sgRNA in the FASTQ files.
FASTQ-splitter.pl - a perl script for use after the library has been amplified and submitted to next-generation sequencing. If the exact primers described in the original publication (with 9x variable length spacers on the F read, and 4 barcodes at the other end) are used to generate the Illumina product, this script will automaticallly split the resulting FASTQ file into separate files for each barcode.
library-counts.txt - A description of the genes targeted, the sgRNA sequence, and the number of counts in the depositor's Illumina sequencing of the original library
sgRNA-library.out - The list of all sgRNAs present in the library
sgRNA-library-cloned.bed - A BED file that describes the position of all of the sgRNAs in the library, that can be uploaded to e.g. the UCSC genome browser to look at the positions of the sgRNAs.
test-guides.txt - a text file with a few test sgRNAs
10000-WTCHG-154382-1.fastq - a small FASTQ file to use for testing the perl scripts
To design the sgRNAs, the depositing laboratory computed all possible sgRNA target sites within the shared exonic regions on either DNA strand of the Drosophila genome. These were ranked based on their position relative to the beginning of the coding sequence, since frameshifts early in the coding sequence are likely to have a more detrimental effect on protein function.
To mitigate potential off-target effects, they mapped potential guide RNAs to all possible off-target sites in the Drosophila genome with up to 3 mismatches. Any sgRNAs that mapped to a potential off-target site were excluded, and five non-overlapping guide RNAs were selected for each gene. The cut off of 3 mismatches was chosen because of recent studies in Drosophila, which suggest that off-targets with more than 3 mismatches are highly unlikely to be mutagenized by the Cas9 protein.
These selected sgRNAs, plus common adaptors, were synthesised as a large pool, followed by PCR amplification using the common sequences, and digestion with a restriction enzyme to release the sgRNA sequences. These sequences were purified and cloned into an
S2 expression vector (Addgene #49330)
which expresses the sgRNA from a Drosophila U6:2 promoter, and the Cas9 protein with N- and C-terminal nuclear localisation signals under the control of the actin 5C promoter. Additionally, the vector contains a puromycin N-acetyltransferase gene to allow selection in S2 cells.
Cloning of the sgRNAs uses the type IIS restriction enzyme BspQI that allows scarless integration of the 20nt target sequence. In order to maintain representation of the library, a total of approximately 7 million bacterial colonies were generated, representing at least a 100-fold coverage of the library.
To assess the coverage of sgRNAs in the library, the Liu lab performed high throughput sequencing (HTS) of a PCR product across the sgRNA sequences, at a depth of 827,527 mapped reads.
This showed that 40,279 sgRNAs were represented by at least one sequencing read and 21,805 sgRNAs by more than 5 reads. Analysis of the genes represented in the library showed that 13,501 genes (98.8%) were represented by at least one sgRNA and 8989 genes (65.8%) were targeted by 3 or more sgRNAs. This is of particular note since recent data suggest that the specificity of sgRNA screens can be improved by selecting those genes where similar effects are seen for multiple independent sgRNAs.
BED files of both the total 68,340 sgRNA set and cloned 40,279 sgRNA set are available, which can be uploaded directly to a genome browser.
These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the pooled libraries were described, and include Addgene in the Materials and Methods of your future publications.
Example for your Materials & Methods section:
Oxford Drosophila CRISPR Pooled Library was a gift from Ji-Long Liu (Addgene #64750)
For your References section:
Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9 .
Bassett AR, Tibbit C, Ponting CP, Liu JL. Biol Open. 2013 Dec 10. pii: bio.20137120v1. doi: 10.1242/bio.20137120.PubMed 26165496