Functional genomics using CRISPR-CAS systems for saturating mutagenesis of non-coding elements, compositions, methods, libraries and applications thereof

An isolated modified eukaryotic cell obtained by a method comprising

• introducing a deep scanning mutagenesis guide library into a population of cells for screening non-coding genomic sites associated with a change in a phenotype, the population of cells comprising a plurality of Type II CRISPR-Cas system guide molecules,
• wherein each cell of the population contains a Cas polypeptide and no more than one guide molecule,
• wherein each guide molecule targets a non-coding genomic sequence to generate an indel mutation within a continuous genomic region,
• wherein the indel mutation has a mean deletion size of 11 bp and a mean insertion size of 4 bp that is offset by about 10 bp from an expected cleavage point,
• wherein the plurality of guide molecules target at least 100 genomic non-coding sequences comprising non-overlapping cleavage sites upstream of a PAM sequence for every 1000 base pairs within the at least one continuous genomic region;
• wherein the plurality of guide molecules are capable of targeting two or more continuous genomic regions that physically interact, the at least one continuous genomic region comprising noncoding sequences,
• wherein the guide molecules have a median adjacent genomic cleavage distance between 4 bp and 20 bp, and
• sorting the cells into at least two groups based on the change in phenotype;
• and
• determining relative representation of the guide molecules present in each group, whereby genomic sites associated with the change in phenotype are determined by the representation of guide molecules present in each group;
• preparing a Type II CRISPR-Cas composition comprising the one or more guide molecules identified in the library that target the non-coding genomic sites determined to be associated with the change in phenotype;
• administering the Type II CRISPR-Cas composition to an unmodified eukaryotic cell to obtain the modified eukaryotic cell comprising one or more single-site indel mutations at the non-coding genomic site determined to be associated with the change in phenotype,
• wherein the genomic sites associated with the change in phenotype comprise one or more transcription enhancer or repressor elements.