Methods and compositions for altering function and structure of chromatin loops and/or domains

Patent metadata

Field	Value
Publication number	US-2018245079-A1
Application number	US-201615753318-A
Country	US
Kind code	A1
Filing date	Aug 18, 2016
Priority date	Aug 18, 2015
Publication date	Aug 30, 2018
Grant date	—

Abstract

Chromatin 3D structure modulating agents in the context of the present invention are intended to interfere or manipulate the function of loop anchor motifs, such as CTCF motifs. In certain example embodiments, the present invention may block formation of an loop anchor or chromatin domain or induce formation of a loop anchor or chromatin domain at a targeted genomic location. For instance, a loop anchor motif can be altered, such as by mutating (including inverting) a binding motif so as to remove such a motif, or by adding new binding motifs in new locations within a loop domain, so as to reduce the size of an existing loop, so as to modify the size of an existing loop, or combinations thereof. Alternatively, the chromatin 3D structure modulating agent may bind a target region and mask a loop anchor motif, thereby preventing a loop anchor or chromatin domain from forming. The chromatin 3D structure modulating agent may bind a target region and cause a loop anchor of chromatin domain to form.

First claim

1 . A method to engineer chromatin loops and contact domains in a target region of chromatin DNA inside the nucleus of a cell, said method comprising the step of interfering with the function of CTCF and/or cohesin during the extrusion process wherein chromatin DNA is extruded by each of the two subunit of a CTCF and/or cohesin-comprising extrusion complex in opposite direction with respect to the genome and halted by a forward and reverse CTCF or cohesin binding motif in convergent orientation on opposite strands of the extruded chromatin DNA.

2 . The method of claim 1 , wherein said interfering comprises removal of one or more existing chromatin loops or contact domains, the introduction of one or more new chromatin loops or contact domains, or the modification of one or more existing loops or contact domains,
wherein the removal of one or more existing chromatin loops or contact domains comprises:
i. the targeted removal or modification of one or more existing forward and/or reverse CTCF or cohesin binding motifs in or proximate to said target region; or
ii. the targeted removal or modification of one or more, preferably all, CTCF or cohesin binding motifs located at the contact domain boundary,
wherein the introduction of one or more new chromatin loops or contact domains comprises:
i. the targeted introduction of one or more new forward and/or reverse CTCF or cohesin binding motifs in or proximate to said target region; or
ii. the targeted introduction of one or more new forward and/or reverse CTCF or cohesin binding motifs in or proximate to said target region to thereby create two consecutive CTCF or cohesin binding motifs that do not loop to one another, and/or
wherein the modification of one or more existing loops or contact domains comprises:
i. the targeted introduction of one or more new forward and/or reverse CTCF or cohesin binding motifs; or
ii. the targeted introduction of one or more extrusion-blocking proteins or protein-binding sites in or proximate to said target region to thereby prevent the extrusion of at least one chromatin strand through the extrusion complex whereby a smaller loop is form or a loop is blocked from forming, preferably said introduction being in a location between the forward and reverse CTCF or cohesin binding motifs at an existing loop or contact domain boundary, more preferably in a location within 1000 base pairs following an existing forward CTCF or cohesin binding motif.

3 . (canceled)

4 . (canceled)

5 . (canceled)

6 . (canceled)

7 . (canceled)

8 . (canceled)

9 . The method of claim 2 , wherein said targeted removal or modification comprises the mutation or inversion of said one or more CTCF or cohesin binding motifs, preferably wherein said targeted removal or modification comprises the mutation of at least a single base pair in said one or more CTCF binding motifs, and/or
wherein said targeted introduction comprises the introduction of one or more CTCF or cohesin binding motifs, preferably in convergent orientation on opposite strands of the chromatin DNA.

10 . (canceled)

11 . The method of claim 2 , wherein said targeted removal, modification or introduction comprises:
i. genome editing; and/or ii. the use of a CRISPR-Cas system, an inactivated CRISPR-Cas system, a Cas protein, a zinc finger protein (ZFP), a zinc finger nuclease (ZFN), a transcription activator-like effector (TALE), a transcription activator-like effector nuclease (TALEN), or a meganuclease.

12 . (canceled)

13 . The method of claim 1 , wherein the CTCF or cohesin binding motif is the CTCF motif, and/or
wherein said contact domain is an exclusion domain, and wherein said exclusion domain is introduced by inserting, or deleted by deleting, a CTCF or cohesin binding motif downstream or upstream from adjacent CTCF or cohesin binding motifs in convergent orientation, and/or wherein the extrusion complex comprises one or more members selected from the group consisting of CTCF, SA1/2, Smc3, Smc1, cohesin and Rad21, and/or wherein said target region comprises genes the expression of which is to be modified, preferably wherein said proximity to the target region is less than 1000 base pairs, and/or wherein said target region is located in or overlaps with an existing chromatin loop or contact domain, or wherein said target region is to be formed into or is to be made part of a new chromatin loop or contact domain.

14 . (canceled)

15 . The method according to claim 1 , wherein in addition to the step of interfering with the function of CTCF and/or cohesin, said method comprises the step of performing in situ Hi-C on said cell prior to or following said step of interfering with the function of CTCF and/or cohesin, optionally combined with HYbrid Capture on the in situ Hi-C library generated, or
wherein in addition to the step of interfering with the function of CTCF and/or cohesin, said method comprises the step of performing in situ Hi-C on said cell prior to or following said step of interfering with the function of CTCF and/or cohesin, optionally combined with HYbrid Capture on the in situ Hi-C library generated, wherein said in situ HiC method identifies target chromatin loop modification sites or monitors the result of chromatin loop or contact domain modification in a target region, said method comprising performing prior to or following said step of interfering with the function of CTCF and/or cohesin the steps of: a. generating a 3D contact map of the genome of said cell; b. identifying a target modification site from the 3D contact map, wherein the target modification site comprises either an existing loop or domain or a target nucleic acid sequence for introducing a new chromatin loop or domain, or identifying modified sites from the 3D contact map, wherein a modified site comprises a modified loop or domain, optionally wherein said method further comprises the steps of: c. generating a set of vectors wherein each vector encodes one or more chromatin loop perturbations, wherein expression of the one or more vectors results in removal of one or more existing chromatin loops or domains, introduction of one or more new chromatin loops or domains, or modification of one or more existing loops or domains at one of the identified target modification sites; d. delivering each vector in the set of vectors to a different cell or cell population to determine an impact of the introduced chromatin loop perturbations on cell function; and e. identifying one or more vectors that introduce the one or more chromatin perturbations with a minimal negative impact on cell function.

16 . The method of claim 1 , wherein said method is for altering chromatin three dimensional (3D) structure in a cell, and/or
wherein said method comprises delivering to the nucleus of a cell one or more sequence-specific DNA targeting agents directed to said target region or proximate thereto, preferably wherein said one or more sequence-specific DNA targeting agents are selected from the group consisting of a CRISPR-Cas system, a Cas protein, a catalytically inactive CRISPR-Cas system or Cas protein, a zinc finger protein (ZFP), a zinc finger nuclease (ZFN), a transcription activator-like effector (TALE), a transcription activator-like effector nuclease (TALEN), and a meganuclease.

17 . (canceled)

18 . (canceled)

19 . (canceled)

20 . The method of claim 16 , wherein delivering the one or more sequence-specific DNA targeting agents to the nucleus of a cell comprises:
i. delivering one or more vectors

Assignees

• Broad Inst Inc
• Baylor College Medicine

Inventors

• Lieberman Aiden Erez
• Lander Eric S
• Rao Suhas
• Huang Su-Chen
• Sanborn Adrian L
• Durand Neva C
• Huntley Miriam
• Jewett Andrew

Classifications

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C07K14/47 Primary

  from mammals · CPC title