![]() In some cases, the small guide RNA has 19-25 or 21-28 nucleotides between the unique endonuclease site and the 5′ end. In some cases, the unique endonuclease cut site is a BlpI site. In some cases, the 5′ hairpin region comprises fewer than four consecutive uracil nucleotides and a length of at least 31 nucleotides. In some cases, the plurality of cells are contacted with a library of structurally distinct short hairpin RNAs (shRNA). In some cases, the plurality of cells comprise a tetracycline transactivator, and wherein the method comprises expression of dCas9 under the control of a tetracycline inducible promoter in the absence of tetracycline or other exogenous inducer of the tetracycline inducible promoter. In some cases, the promoter operably linked to the polynucleotide encoding the dCas9 is inducible. In some cases, the nuclease deficient sgRNA-mediated nuclease (dCas9) is encoded by an expression cassette in the cell, the expression cassette comprising a promoter operably linked to a polynucleotide encoding the dCas9. In some cases, the promoter operably linked to the polynucleotide encoding the sgRNA is inducible. In some cases, the sgRNA is encoded by an expression cassette in the cell, the expression cassette comprising a promoter operably linked to a polynucleotide encoding the sgRNA. In some cases, the selecting the cells on the basis of the phenotype comprises fluorescence activated cell sorting, affinity purification of cells, or selection based on cell motility. ![]() In some cases, the selecting the cells on the basis of the phenotype comprises selecting the cells on the basis of protein expression, RNA expression, or protein activity. In some cases, the selection agent is a chemotherapeutic, a DNA damaging agent, a cytotoxic agent, a growth factor, a transcription factor, a kinase, a drug, an exogenous gene under the control of a heterologous promoter, or a hormone. In some cases, the culturing is performed in the presence of a selection agent. In some cases, the selecting the cells on the basis of the phenotype comprises culturing the cells, thereby selecting the cells on the basis of cellular proliferation. In some cases, the deep sequencing comprises sequencing with a redundancy of at least about 10. In some cases, the quantitating the frequency comprises deep sequencing. Binding of the CRISPR:Cas complex to the target sequence results in double stranded cleavage of the target sequence. The mechanism underlying this immunity is based on sequence specific cleavage of foreign nucleic acids by a CRISPR:Cas complex that contains a guide RNA that provides target sequence specificity through a single stranded binding region and is derived from the CRISPR sequences and a guide RNA dependent nuclease encoded by the Cas gene. In Streptococcus thermophilus and Escherichia coli, CRISPR/Cas loci have been demonstrated to confer immunity against bacteriophage infection by an interference mechanism that relies on the strict identity between CRISPR spacers and phage target sequences. They are generally flanked by a set of CRISPR-associated (Cas) genes that encode a nuclease that is important for CRISPR maintenance and function. Clustered, regularly interspaced short palindromic repeat (CRISPR) sequences are present in approximately 40% of eubacterial genomes and nearly all archaeal genomes sequenced to date, and consist of short (24-48 nucleotide) direct repeats separated by similarly sized, unique spacers.
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