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US 10,851,369 B2Granted
Frequency-based modulation of diverse species in a nucleic acid library
AssigneePresident and Fellows of Harvard College
InventorsReza Kalhor, Javier Fernandez Juarez, Henry Hung-yi Lee, George M. Church
Life of this patent
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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.
Claims32
A method of processing a collection of nucleic acid sequences comprising
- connecting an adaptor to one or more members of the nucleic acid sequences in the collection to create a processed nucleic acid template library, wherein the adaptor includes a first DNA sequence encoding a protospacer adjacent motif (PAM) sequence, a tracr mate sequence and a tracrRNA sequence, forming a sequence encoding a guide RNA scaffold sequence
- amplifying members of the processed nucleic acid template library in a polymerase chain reaction (PCR), wherein a thermophilic Cas nuclease and a thermophilic RNA polymerase are added to the PCR reaction, and where in each amplification cycle, the RNA polymerase transcribes a guide RNA from members of the nucleic acid template library and Cas nuclease forms a colocalization complex with transcribed guide RNAs and target amplicons corresponding to guide RNAs, and wherein the Cas nuclease cuts the target amplicon to reduce amplification bias in the library of DNA amplicons to create an adjusted amplified library.
A method of reducing amplification bias in a library of DNA amplicons comprising
- connecting a promoter sequence and a first DNA sequence encoding a PAM sequence, a tracr mate sequence and a tracrRNA sequence to each member of a DNA library to create a processed DNA template library, wherein a portion of the member of the DNA library, the PAM sequence, the tracr mate sequence and the tracrRNA sequence form a self-targeting or homing guide RNA template sequence, and
- amplifying members of the processed DNA template library in a polymerase chain reaction (PCR), wherein a thermophilic Cas nuclease and a thermophilic RNA polymerase are added to the PCR reaction, and where in each amplification cycle, the RNA polymerase transcribes a guide RNA from members of the DNA template library and Cas nuclease forms a colocalization complex with transcribed guide RNAs and target amplicons corresponding to guide RNAs, and wherein the Cas nuclease cuts the target amplicon to reduce amplification bias in the library of DNA amplicons to create an adjusted amplified library.
Dependencies are inferred from the claim text.