Abstract: The present disclosure is directed, in some embodiments, to compositions and methods for inducible modification of a cell genome.

Abstract: The present disclosure provides a non-naturally occurring CRISPR-Cas system comprising: a Cas9 effector protein capable of generating cohesive ends (stiCas9), and a guide polynucleotide that forms a complex with the stiCas9 and comprising a guide sequence, wherein the guide sequence hybridizes with a target sequence in a eukaryotic cell but does not hybridize to a sequence in a bacterial cell, and wherein the complex does not occur in nature. The present disclosure also provides a method of introducing a sequence of interest into a chromosome of a cell. Finally, the present disclosure provides for a method of modifying one or more nucleotides using seamless mutagenesis.

Abstract: The present disclosure provides methods of inserting a polynucleotide of interest into the genome of a eukaryotic cell, wherein said methods comprise improving the efficiency of CRISPR/Cas-mediated polynucleotide insertion by addition of an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway to the eukaryotic cell. The present disclosure further provides compositions for inserting a polynucleotide of interest into the genome of a eukaryotic cell, and kits for inserting a gene of interest into the genome of a eukaryotic cell.

Abstract: Provided are Cas9 effector proteins having enhanced stability. Embodiments of the Cas9 effector proteins have a first nuclear localization signal attached to the N-terminus and a second nuclear localization signal attached to the C-terminus. Also provided are Cas9 systems comprising Cas9 effector proteins having enhanced stability and a guide polynucleotide that forms a complex with the Cas9 effector protein. Further provided are methods for providing site-specific modification of a target sequence in a eukaryotic cell using the Cas9 effector proteins.

Abstract: The present disclosure provides a polynucleotide comprising an RNA guide sequence, a Cas-binding region, and a DNA template sequence. The disclosure also provides compositions comprising a Cas nuclease or a Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, and a DNA template sequence. The disclosure further provides a fusion protein comprising a Cas nuclease or a Cas nickase and a DNA polymerase recruitment moiety. Also provided are methods for providing a targeted insertion in a target DNA of a cell.

Abstract: The present disclosure provides proteins, compositions, methods, and kits for improved gene editing efficiency. In some embodiments, the disclosure provides a fusion protein comprising a Cas nuclease and a reverse transcriptase, a DNA polymerase, a DNA ligase, or a combination thereof.

Abstract: The present disclosure provides methods of introducing site-specific mutations in a target cell and methods of determining efficacy of enzymes capable of introducing site-specific mutations. The present disclosure also provides methods of providing a bi-allelic sequence integration, methods of integrating of a sequence of interest into a locus in a genome of a cell, and methods of introducing a stable episomal vector in a cell. The present disclosure further provides methods of generating a human cell that is resistant to diphtheria toxin.

Abstract: Provided herein, in some embodiments, are nucleic acid-based tools that may be used for high-throughput functional genomics studies as well as for the generation of knockout (gene inactivation or deletion) or knockin (gene activation or insertion) cell lines. Tools of the present disclosure include an “activatable reporter cassette,” a guide RNA construct and a nuclease that can be used together, for example, to modify and isolate targeted cells of interest.

Abstract: The present disclosure is directed, in some embodiments, to compositions and methods for inducible modification of a cell genome.

Abstract: The present disclosure provides a non-naturally occurring CRISPR-Cas system comprising: a Cas9 effector protein capable of generating cohesive ends (stiCas9), and a guide polynucleotide that forms a complex with the stiCas9 and comprising a guide sequence, wherein the guide sequence hybridizes with a target sequence in a eukaryotic cell but does not hybridize to a sequence in a bacterial cell, and wherein the complex does not occur in nature. The present disclosure also provides a method of introducing a sequence of interest into a chromosome of a cell. Finally, the present disclosure provides for a method of modifying one or more nucleotides using seamless mutagenesis.

Abstract: The present disclosure is directed, in some embodiments, to compositions and methods for inducible modification of a cell genome.

Abstract: Provided herein, in some embodiments, are nucleic acid-based tools that may be used for high-throughput functional genomics studies as well as for the generation of knockout (gene inactivation or deletion) or knockin (gene activation or insertion) cell lines. Tools of the present disclosure include an “activatable reporter cassette,” a guide RNA construct and a nuclease that can be used together, for example, to modify and isolate targeted cells of interest.

Abstract: The present disclosure is directed, in some embodiments, to compositions and methods for inducible modification of a cell genome.

Abstract: The present invention relates to genetic techniques employing the direct ligatation of an external DNA fragment generated in situ by the same ZFNs that target the genome. ObLiGaRe, i.e., the obligated ligation-gated recombination, is a new method for genetic engineering using custom designed nucleases, and a strategy of site-specific gene insertion utilizing the NHEJ pathway. It applies a similar logic to the one used in unidirectional loxP sites (Oberdoerffer et al., 2003) but maintains all the advantages and flexibility of CDNs.

Abstract: The present invention relates to genetic techniques employing the direct ligatation of an external DNA fragment generated in situ by the same ZFNs that target the genome. ObLiGaRe, i.e., the obligated ligation-gated recombination, is a new method for genetic engineering using custom designed nucleases, and a strategy of site-specific gene insertion utilizing the NHEJ pathway. It applies a similar logic to the one used in unidirectional loxP sites(Oberdoerffer et al., 2003) but maintains all the advantages and flexibility of CDNs.

Abstract: The invention provides a method for inserting a single stranded replacement nucleic acid into a target nucleic acid, the method comprising the steps of: a) generating a single stranded replacement nucleic acid from a double stranded nucleic acid, wherein the double stranded nucleic acid is adapted at one or both of its 5? ends such that preferential degradation of one strand and/or strand separation generates the single stranded replacement nucleic acid, wherein the single stranded replacement nucleic acid comprises a 5? region that is identical to sequence on the target nucleic acid, a 3? region that is identical to sequence on the target nucleic acid and optionally a replacement region between the 5? and 3? regions that is not identical to sequence on the target nucleic acid, b) exposing the target nucleic acid to the single stranded replacement nucleic acid under conditions suitable for recombination to occur between the single stranded replacement nucleic acid and the target nucleic acid, and c) selecting

Abstract: The invention provides a method for inserting a single stranded replacement nucleic acid into a target nucleic acid, said method comprising the steps of: a) generating a single stranded replacement nucleic acid from a double stranded nucleic acid, wherein the double stranded nucleic acid is adapted at one or both of its 5? ends such that preferential degradation of one strand and/or strand separation generates the single stranded replacement nucleic acid, wherein the single stranded replacement nucleic acid comprises a 5? region that is identical to sequence on the target nucleic acid, a 3? region that is identical to sequence on the target nucleic acid and optionally a replacement region between the 5? and 3? regions that is not identical to sequence on the target N nucleic acid, b) exposing the target nucleic acid to the single stranded replacement nucleic acid under conditions suitable for recombination to occur between the single stranded replacement nucleic acid and the target nucleic acid, and c) select