US 12,548,638 B2Granted

Methods for designing guide sequences for guided nucleases

AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology, President and Fellows of Harvard College, New York Genome Center, Inc., New York University

InventorsNeville Sanjana, Feng Zhang, Joshua A. Meier

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Abstract

Embodiments disclosed herein provide methods, including computer-implemented methods, for designing guide sequence which may be incorporated into custom, large scale guide sequence libraries. The methods require only a list of target genes as input and utilize on target and off target scores to generate an optimal set of guide sequences for a set of target genes. In certain embodiments, the methods may also utilize multi-tissue RNA-sequencing data and/or protein annotation to design targets to genes that are highly expressed and/or contain a functional protein domain. The invention further comprises guide libraries, cells comprising said guide libraries. Computer-implemented embodiments further improve computer system function by reducing excessive user wait time through the use of data structures that reduce search from linear to logarithmic time.

Claims15

Dependent claims

A computer-implemented method for making guide sequences comprising:

identifying, using one or more computing devices, exons in one or more target genes, and for each exon identifying potential target regions that can be targeted using a CRISPR-Cas system with an exon prediction module;
ranking, by the one or more computing devices, each potential target region based at least in part by an off-target avoidance score, and an on-target efficiency score determined using a classifier, wherein the off-target avoidance score is determined at least in part by a cutting frequency determination score;
for each target region above a defined cut-off value, generating, by the one or more computing devices, a set of guide sequences that are complementary to the target regions;
and
synthesizing a set of guide molecules comprising the set of guide sequences each guide molecule thereby capable of complexing with a CRISPR-Cas system and directing sequence-specific binding of the complex to a corresponding target region.

2 more elements

Dependencies are inferred from the claim text.

Facts

Application: 16/621,085Filed 6 Jul 2018 · us provisional application
Priority: 7 Jul 2017
Published: US 2020/0202981 A125 Jun 2020
Granted: 10 Feb 2026
Term ends: Not estimatedThe source records don’t support an estimate.

Classification13 codes

C12N 15/88
Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation: using microencapsulation, e.g. using…
G16B 35/10
ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides: design of libraries
C12N 15/1089
Isolating an individual clone by screening libraries: design, preparation, screening or analysis of libraries using computer algorithms
C12N 15/111
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: general methods applicable to biologically…