Granted Patent B2

US 12,406,749 · App. 16/772,747 · Granted Sep 2, 2025

Inventors: Max Walt Shen (Cambridge, MA); Jonathan Yee-Ting Hsu (Cambridge, MA); Mandana Arbab (Cambridge, MA); David K. Gifford (Cambridge, MA); David R. Liu (Cambridge, MA); Richard Irving Sherwood (Cambridge, MA)

Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology; The Brigham and Women's Hospital, Inc.; President and Fellows of Harvard College

G16B20/30A61K31/7088A61K38/465C12N9/22C12N15/1089C12N15/11G16B40/20C12N2310/20C12N2800/80

Abstract

Claims (36)

1. A method of introducing a genetic change into a target genomic location encoding a pathogenic allele to modify the pathogenic allele to become a non-pathogenic allele using a Cas-based double strand break genome editing system, the method comprising:

using a computer hardware processor to perform:

selecting a guide RNA for use in introducing the genetic change into the target genomic location by analyzing inputs indicating a nucleotide sequence of the target genomic location and one or more available cut sites for the Cas-based double strand break genome editing system, the selecting comprising:

(a) determining a microhomology score matrix using a first neural network, the determining comprising:

determining a plurality of pairs of overhang sequences using the inputs;

determining a microhomology length vector and/or a microhomology GC fraction vector using the inputs; and

applying the first neural network to the plurality of pairs of overhang sequences and the microhomology length vector and/or the microhomology GC fraction vector to obtain the microhomology score matrix;

(b) determining a microhomology-independent score matrix using a second neural network, the determining comprising:

determining a deletion length vector using the inputs and the plurality of pairs of overhang sequences; and

applying the second neural network to the deletion length vector to obtain the microhomology-independent score matrix;

(c) determining a probability distribution over 1-bp insertions;

(d) determining, using the microhomology score matrix, the microhomology-independent score matrix and the probability distribution over 1-bp insertion, a probability distribution over indel genotypes and a probability distribution over indel lengths for the nucleotide sequence of the target genomic location and the one or more available cut sites;

(e) determining, using the probability distribution over indel genotypes and the probability distribution over indel lengths, for each guide RNA of a plurality of guide RNAs, a predicted frequency of introducing the genetic change into the target genomic location using the Cas-based double strand break genome editing system and the guide RNA;

(f) selecting, using the predicted frequencies of (e), a guide RNA of the plurality of guide RNAs for use in introducing the genetic change into the target genomic location using the Cas-based double strand break genome editing system; and

introducing the genetic change into the target genomic location using the guide RNA selected at (f) and the Cas-based double strand break genome editing system, wherein the genetic change is a 1 base pair insertion or a 1−60 base pair deletion that modifies the pathogenic allele to become the non-pathogenic allele.

2. The method of claim 1 , wherein analyzing inputs comprises identifying, for a cut site of the one or more available cut sites, a plurality of pairs of overhang sequences, each pair of the plurality of pairs of overhang sequences comprising:

(i) a 3′-overhang sequence; and

(ii) a 5′-overhang sequence.

3. The method of claim 1 , wherein determining the probability distribution over 1-bp insertion comprises:

determining, using the inputs, an overall deletion score, a precision score, and/or cut site nucleotides; and

applying a third neural network to the overall deletion score, the precision score, and/or the cut site nucleotides to obtain the probability distribution over 1-bp insertion.

4. The method of claim 1 , comprising:

contacting a plurality of cells with the guide RNA and the Cas-based double strand break genome editing system; and

introducing, using the selected guide RNA and the Cas-based double strand break genome editing system, the genetic change into the target genomic location of at least 30% of cells of the plurality of cells.

5. The method of claim 1 , comprising:

contacting a plurality of cells with the guide RNA and the Cas-based double strand break genome editing system; and

introducing, using the selected guide RNA and the Cas-based double strand break genome editing system, the genetic change into the target genomic location of at least 50% of cells of the plurality of cells.

6. The method of claim 1 , wherein the Cas-based double strand break genome editing system comprises a type II Cas RNA-guided endonuclease, or a functional variant or ortholog thereof.

7. The method of claim 1 , wherein the Cas-based double strand break genome editing system comprises a Cas9 RNA-guided endonuclease, or a variant or orthologue thereof.

8. The method of claim 7 , wherein the Cas9 RNA-guided endonuclease is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus pyogenes Cas9 (SpCas9), Staphyloccocus aureus Cas (SaCas9), Francisella novicida Cas9 (FnCas9), or a functional variant or orthologue thereof.

9. The method of claim 1 , further comprising:

treating a genetic disease in a subject caused by a pathogenic allele in a genome of a cell of the subject by contacting the genome of the cell of the subject with the selected guide RNA and the Cas-based double strand break genome editing system, in order to correct the pathogenic allele into a non-pathogenic allele in the genome of the cell.

10. The method of claim 1 , wherein selecting the guide RNA for use in introducing the genetic change into the target genomic location using the Cas-based double strand break genome editing system comprises selecting a guide RNA that corresponds to a cut site of the target genomic location.

11. The method of claim 1 , wherein selecting the guide RNA comprises selecting a guide RNA that has a predicted frequency of introducing the genetic change into the target genomic location of at least 30%.

12. The method of claim 1 , wherein selecting the guide RNA comprises selecting a guide RNA that has a predicted frequency of introducing the genetic change into the target genomic location of at least 50%.

13. The method of claim 1 , wherein the genetic change is a 1 base pair insertion or a 1-60 base pair deletion that modifies the pathogenic allele to become a wildtype allele.

Assignments (8)

Continuity (3)

References Cited (400)

Cited By (3)

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