Integrated DNA Technologies, Inc.

This invention pertains to mutant Lachnospiraceae bacterium ND2006 (Lb) Cas12a nucleic acids and proteins for use in CRISPR/Cas12a endonuclease systems, and their methods of use. In particular, the invention pertains to an isolated mutant LbCas12a protein, wherein the isolated mutant LbCas12a protein is active in a CRISPR/Cas12a endonuclease system. The invention also includes isolated nucleic acids encoding mutant LbCas12a proteins, ribonucleoprotein complexes and CRISPR/Cas12a endonuclease systems having mutant LbCas12a proteins.

Described herein is a system and process for identifying and characterizing double-stranded DNA break repair sites that are based on biological information and have improved accuracy. Also described is a sequence alignment process that uses biological data to inform the alignment matrix for position specific alignment scoring, resulting in the identification of noncanonical target sites.

The present invention is directed to methods and compositions for adding tails of specific lengths to a substrate polynucleotide. The invention also contemplates methods and compositions for immobilization of tailed substrates to a solid support. The disclosure contemplates that the attenuator molecule is any biomolecule that associates with a tail sequence added to a substrate polynucleotide and controls the addition of a tail sequence to the 3′ end of the substrate polynucleotide. The sequence that is added to the substrate polynucleotide is referred to herein as a tail sequence, or simply a tail, and the process of adding a nucleotide to a substrate polynucleotide is referred to herein as tailing.

Described herein are methods and compositions for improved hybridization capture for enriching for target nucleic acid sequences from a population of nucleic acids. In one aspect, the methods and compositions are useful for Next Generation Sequencing (NGS) applications. The methods and compositions include combining in solution capture probes and target nucleic acid and permitting hybridization of the target nucleic acid to the capture probes under conditions to promote efficient hybridization. Following hybridization, the probe/target complex is immobilized to a capture material while simultaneously incubating at an optimum hybridization temperature. The incubation denatures unwanted nucleic acids from the capture probes further enriching for target nucleic acid.

The invention can be used to provide a more efficient and less error-prone method of detecting variants in DNA, such as SNPs and indels. The invention also provides a method for performing inexpensive multiplex assays. The invention also provides methods for detection of DNA sequences altered after cleavage by a targetable endonuclease, such as the CRISPR Cas9 protein from the bacterium Streptococcus pyogenes.

This invention pertains to mutant Cas9 nucleic acids and proteins for use in CRISPR/Cas endonuclease systems, and their methods of use. In particular, the invention pertains to an isolated mutant Cas9 protein, wherein the isolated mutant Cas9 protein is active in a CRISPR/Cas endonuclease system, wherein the CRISPR/Cas endonuclease system displays reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system. The invention also includes isolated nucleic acids encoding mutant Cas9 proteins, ribonucleoprotein complexes and CRISPR/Cas endonuclease systems having mutant Cas9 proteins that display reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system.

This invention pertains to optimized protein fusion linkers for creating multi-functional chimeric proteins and methods of using the same. Additionally, the invention pertains to chimeric proteins for use in guided endonuclease systems.

Described herein is a series of recombinantly engineered Cas12a proteins from Eubacterium rectale . In one aspect, various mutations to the wild type Eubacterium rectale Cas12a polypeptide (ERCAS12A) are provided that have enhanced activity.

Described herein are CAS12A mutants from Lachnospiraceae bacterium and methods for use thereof. These mutants have enhanced DNA cleavage activities at non-canonical TTTT protospacer adjacent motifs (PAM) compared to the wild-type enzyme.

Described herein are methods and compositions for improved hybridization capture for enriching for target nucleic acid sequences from a population of nucleic acids. In one aspect, the methods and compositions are useful for Next Generation Sequencing (NGS) applications. The methods and compositions include combining in solution capture probes and target nucleic acid and permitting hybridization of the target nucleic acid to the capture probes under conditions to promote efficient hybridization. Following hybridization, the probe/target complex is immobilized to a capture material while simultaneously incubating at an optimum hybridization temperature. The incubation denatures unwanted nucleic acids from the capture probes further enriching for target nucleic acid.