Abstract: Provided herein are compositions comprising a CRISPR array comprising one or more crRNA sequences and a 5? direct repeat (DR) sequence linked to a barcode guide RNA (bcgRNA), wherein the bcgRNA comprises from 5? to 3? (a) a barcode sequence, and (b) a reverse-transcription handle. Also provided are methods that comprise using the described CRISPR arrays to introduce one or perturbations in a single cell transcriptome.

Abstract: The application relates to a deep scanning mutagenesis library to interrogate phenotypic changes in a population of cells comprising a plurality of CRISPR-Cas system guide RNAs targeting genomic sequences within at least one continuous genomic region, wherein the guide RNAs target at least 100 genomic sequences upstream of a PAM sequence for every 1000 base pairs within the continuous genomic region and methods for their use.

Abstract: Provided herein are compositions and methods comprising modified crRNA comprising a spacer sequence and a direct repeat sequence, wherein the crRNA comprises one or more chemically modified nucleotides. Also provided are methods of enhancing modulation of gene transcripts in a cell, the method comprising introducing into the cell a modified crRNA as described herein, and a Cas13 polypeptide, an mRNA encoding a Cas13 polypeptide, and/or a recombinant expression vector comprising a nucleotide sequence encoding a Cas13 polypeptide, wherein the modified crRNA guides the Cas13 polypeptide to the target RNA sequence, and wherein the modified crRNA induces regulation of the target RNA with an enhanced activity relative to a corresponding unmodified crRNA.

Abstract: A Class 2, Type VI clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) which comprises a direct repeat (DR) stem loop sequence and a guide or spacer sequence, is provided characterized by a DR selected from those of Table 9. Also described is are methods for generating, selecting, characterizing and optimizing a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) for use in the CRISPR-Cas13d system described herein. Also provided is a screening method to identify crRNA particularly suited for use with specified targets. Further, the invention includes non-naturally occurring, synthesized or engineered crRNAs as described herein along with nucleic acid molecule, vectors, RNPs, cells, libraries, and compositions comprising the same, and uses thereof in treating a disease or in functionally screening a gene.

Abstract: An in vitro method is provided for analyzing chromatin accessibility and screening RNA of each single cell in a heterologous population (e.g., a library of cells). The method comprises incubating cell nuclei obtained from lysed cells with a transposome complex in a tagmentation buffer, performing reverse transcription wherein each of the RNAs is reverse transcribed to a DNA barcoded with the first barcode; sequencing DNA, which is extracted from digested cell nuclei; and analyzing chromatin accessibility and RNA of the cells. In a further embodiment, the method described comprises performing combinatorial cellular indexing and/or a perturbation step. Additionally, provided are a transposase TnY, buffer(s), and kit(s) for use in the described method.

Abstract: Provided herein is a recombinant or engineered Cas9 protein. The Cas9 protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. The Cas9 protein has at least one mutation in an amino acid residue selected from 262, 324, 409, 480, 543, 694, of the amino acid sequence provided in SEQ ID NO: 2 or the corresponding residue of an aligned sequence, and at least one mutation in an amino acid residue selected from 1111, 1135, 1218, 1219, 1322, 1335, and 1337, of the amino acid sequence provided in SEQ ID NO: 2 or the corresponding residue of an aligned sequence. The amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of a naturally occurring Cas9 protein.