公开(公告)号：US10851369B2

公开(公告)日：2020-12-01

申请号：US16310643

申请日：2017-06-21

Applicant: President and Fellows of Harvard College

Inventor： Reza Kalhor ,  Javier Fernandez Juarez ,  Henry Hung-yi Lee ,  George M. Church

IPC: C12Q1/68 ,  C12N15/10 ,  C12N15/11 ,  C40B40/02 ,  C40B50/06 ,  C12N15/09 ,  C12N9/22

Abstract: A method of processing a collection of nucleic acid sequences is provided including connecting an adaptor to one or more or each nucleic acid sequence in the collection to create a processed nucleic acid template library, wherein the adaptor includes a first DNA sequence encoding a PAM sequence and at least a tracr mate.

公开(公告)号：US10745814B2

公开(公告)日：2020-08-18

申请号：US16099487

申请日：2017-05-09

Applicant: President and Fellows of Harvard College

Inventor： Reza Kalhor ,  Henry Hung-yi Lee ,  George M. Church

IPC: C25B3/10 ,  C12Q1/6844 ,  C12P19/34 ,  C25B3/02 ,  C25B3/04 ,  C12Q1/6806

Abstract: The present disclosure provides methods of activating an enzyme, such as error prone or template independent polymerase, using electricity to alter pH of a reaction zone and reaction site from an inactivating pH at which the enzyme is inactive to an activating pH at which the enzyme is active to add a nucleotide to an initiator or growing polymer chain. The activating pH can then be changed back to an inactivating pH and the process repeated as many times as desired to produce a target nucleic acid sequence.

公开(公告)号：US20190177720A1

公开(公告)日：2019-06-13

申请号：US16310643

申请日：2017-06-21

Applicant: President and Fellows of Harvard College

Inventor： Reza Kalhor ,  Javier Fernandez Juarez ,  Henry Hung-yi Lee ,  George M. Church

IPC: C12N15/10 ,  C12N15/11 ,  C12N9/22

Abstract: A method of processing a collection of nucleic acid sequences is provided including connecting an adaptor to one or more or each nucleic acid sequence in the collection to create a processed nucleic acid template library, wherein the adaptor includes a first DNA sequence encoding a PAM sequence and at least a tracr mate.

公开(公告)号：US20210108243A1

公开(公告)日：2021-04-15

申请号：US16951254

申请日：2020-11-18

Applicant: President and Fellows of Harvard College

Inventor： Henry Hung-yi Lee ,  George M. Church ,  Reza Kalhor

IPC: C12P19/34 ,  C12Q1/6844

Abstract: The disclosure provides methods for making a polynucleotide wherein the addition of nucleotides can be physically, chemically and/or enzymatically controlled. The methods include combining a selected nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3′ terminal nucleotide, wherein the reaction reagents can be modulated and under conditions that allow covalent addition of one or more of a selected nucleotide to the 3′ terminal nucleotide such that the selected nucleotide becomes a 3′ terminal nucleotide, and repeating the addition step until the polynucleotide is formed.

公开(公告)号：US20190145013A1

公开(公告)日：2019-05-16

申请号：US16099487

申请日：2017-05-09

Applicant: President and Fellows of Harvard College

Inventor： Reza Kalhor ,  Henry Hung-yi Lee ,  George M. Church

IPC: C25B3/10 ,  C12P19/34 ,  C25B3/02 ,  C25B3/04

Abstract: The present disclosure provides methods of activating an enzyme, such as error prone or template independent polymerase, using electricity to alter pH of a reaction zone and reaction site from an inactivating pH at which the enzyme is inactive to an activating pH at which the enzyme is active to add a nucleotide to an initiator or growing polymer chain. The activating pH can then be changed back to an inactivating pH and the process repeated as many times as desired to produce a target nucleic acid sequence.

公开(公告)号：US20190112626A1

公开(公告)日：2019-04-18

申请号：US16090640

申请日：2017-03-30

Applicant: President and Fellows of Harvard College

Inventor： Henry Hung-yi Lee ,  George M. Church ,  Reza Kalhor

IPC: C12P19/34

Abstract: The disclosure provides methods for making a polynucleotide wherein the addition of nucleotides can be physically, chemically and/or enzymatically controlled. The methods include combining a selected nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3′ terminal nucleotide, wherein the reaction reagents can be modulated and under conditions that allow covalent addition of one or more of a selected nucleotide to the 3′ terminal nucleotide such that the selected nucleotide becomes a 3′ terminal nucleotide, and repeating the addition step until the polynucleotide is formed.

公开(公告)号：US11384358B2

公开(公告)日：2022-07-12

申请号：US16498465

申请日：2018-03-29

Applicant: President and Fellows of Harvard College

Inventor： Anik Debnath ,  Javier Fernandez Juarez ,  Henry Hung-yi Lee ,  George M. Church

IPC: C12N15/63 ,  C12N1/20 ,  C12N15/74 ,  C12R1/23 ,  C12R1/24 ,  C12R1/25 ,  C12R1/245

Abstract: The disclosure provides methods of making a tetracycline inducible expression system in a cell. The methods include providing the cell with a first nucleic acid sequence comprising a first promoter operably linked to a tetracycline repressor gene coding sequence, providing the cell with a second nucleic acid sequence comprising a second promoter operably linked to a coding sequence of a gene of interest wherein the second promoter is modified to include one or more tetracycline repressor protein binding sites, and determining the expression of the gene of interest in the presence or absence of tetracycline. The disclosure further provides nucleic acid sequences, vectors and cells including the tetracycline inducible modified promoter.

公开(公告)号：US20190241899A1

公开(公告)日：2019-08-08

申请号：US16339019

申请日：2017-10-05

Applicant: President and Fellows of Harvard College

Inventor： George M. Church ,  Henry Hung-yi Lee ,  Nili Ostrov

IPC: C12N15/74

CPC classification number: C12N15/74 ,  C07K14/005 ,  C12N15/102 ,  C12N15/90 ,  C12N2795/00022 ,  C12N2795/00043 ,  C12Q2521/507

Abstract: Methods and compositions are provided for modulating expression of a target nucleic acid sequence within a non-E. coli cell. The method includes providing the cell with a guide RNA comprising a portion that is complementary to all or a portion of the target nucleic acid sequence, and providing the cell a Cas protein, wherein the guide RNA and the Cas protein co-localize at the target nucleic acid sequence and wherein the Cas protein modulate the expression of the target nucleic acid sequence.