Each construct in this CRISPR\/Cas9 dual library has been designed to simultaneously express two sgRNAs targeting the same gene. The two sgRNAs flank a bi-directional U6 promoter where the 5\u2019 sgRNA is in reverse complement orientation and is driven by a chicken U6 promoter (also in reverse complement orientation), and a human U6 promoter drives expression of the 3\u2019 sgRNA. Each construct also harbors a unique barcode identifier that is flanked by standard Illumina adapter sequences. This allows multiplexed screening samples to be quickly adapted for next generation sequencing with standard Illumina chemistries.<\/p>\n
sgRNA selection criteria for inclusion in the CRISPR\/Cas9 libraries:<\/p>\n
i) sgRNA activity (likelihood of inducing a double strand break)<\/p>\n
a. To predict sgRNA activity, random forest regression was performed on the sgRNA efficacy measurements reported in Doench et al. 2014 and Chari et al. 2015.<\/p>\n
ii) Evolutionary conservation of target site<\/p>\n
a. Conservation scores of sgRNA targets were assigned based on the combined Protein Variation Effect Analyzer scores of the amino acid where the double strand break is likely to occur and its neighboring amino acids.<\/p>\n
iii) The likelihood that Cas9-induced double strand breaks at the target will result in frameshift mutations.<\/p>\n
a. To predict the likelihood that an sgRNA will induce a frameshift mutation, linear regression was performed on the dataset presented in Bae et al. 2014. This algorithm predicts the likelihood of each micro-homology based repair resolution for a given sgRNA target site. The probability of a resolution resulting in a frameshift mutation is then calculated as the sum of out-of-frame resolution \u201cstrengths\u201d divided by the sum of all resolution \u201cstrengths\u201d.<\/p>\n
For each gene the top 20 sgRNAs were selected based on the above criteria. From these 20 sgRNAs, the top 10 that did not align to alternative targets were selected for library inclusion.<\/p>\n
sgRNAs were paired to:<\/p>\n
i) minimize the event of target site overlap<\/p>\n
ii) maximize the likelihood of inducing a frameshift mutation (either individually or due to a deletion between the target site)<\/p>\n
iii) maximize the likelihood that at least one sgRNA targets a conserved sequence.<\/p>\n
The result is an arrayed CRISPR library that contains extremely potent dual sgRNAs that target conserved regions of the genome and have minimal to no off-target effects. The constructs in the library can be pooled for screening where a unique barcode helps with next generation sequencing analysis.<\/p>\n
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Vector: pCLIP-dual-SFFV-ZsGreen-Puromycin (V163)<\/p>\n
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