Systems, methods and compositions for sequence manipulation with optimized functional crispr-cas systems

A non-naturally occurring or engineered composition comprising:

• one or more guide RNA (sgRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell,
• a CRISPR enzyme comprising at least one or more nuclear localization sequences,
• wherein the CRISPR enzyme comprises at least one mutation, such that the CRISPR enzyme has no more than 5% of the nuclease activity of the CRISPR enzyme not having the at least one mutation,
• wherein at least one loop of at least one sgRNA is modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins, and
• wherein the adaptor protein is associated with one or more functional domains.

A modified CRISPR enzyme wherein the enzyme is a Cas9 ortholog of a genus belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter and wherein differences from the corresponding wild type CRISPR enzyme comprise:

• the modified CRISPR enzyme is truncated in comparison to a wild type CRISPR enzyme, or
• the CRISPR enzyme has a length of at least 500 amino acids, at least 800-899 amino acids, at least 900-999 amino acids, at least 1000-1099 amino acids, at least 1100-1199 amino acids, at least 1200-1299 amino acids, at least 1300-1399 amino acids, at least 1400-1499 amino acids, at least 1500-1599 amino acids, at least 1600-1699 amino acids or at least 2000 amino acids, and/or
• the CRISPR enzyme is a nuclease directing cleavage of both strands at the location of the target sequence, or the CRISPR enzyme is a nickase directing cleavage of one strand at the location of the target sequence, and/or
• the CRISPR enzyme is codon-optimized or codon-optimized for expression in a eukaryotic cell, and/or
• the CRISPR enzyme comprises one or more mutations,
• the CRISPR enzyme comprises a chimeric CRISPR enzyme, and/or
• wherein the Cas comprises a helical domain 2 truncation.