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Methods and systems of molecular recording by crispr-cas system

A DNA sequence and cell technology, applied in biochemical equipment and methods, DNA/RNA fragments, DNA preparation, etc., can solve problems such as lack and limitation

Inactive Publication Date: 2018-12-21
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ability to write arbitrary information into DNA, especially within the genome of living cells, is limited by the lack of biologically compatible recording systems that can exploit the full encoding capacity of anything approaching the nucleic acid space

Method used

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  • Methods and systems of molecular recording by crispr-cas system
  • Methods and systems of molecular recording by crispr-cas system

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Experimental program
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Effect test

Embodiment 1

[0077] Materials and methods

[0078] Bacterial Strains and Culture Conditions

[0079] Expression and neospacer collection were performed in BL21-AI cells. Cells were grown at 37°C in Luria Broth (LB) with shaking (240 rpm) unless otherwise stated. L-arabinose (Sigma-Aldrich) (from 20% stock solution in water) was used at 0.2% final concentration (w / w) and isopropyl-β-D-sulfur at 1 mM final concentration Substituted galactopyranoside (IPTG; Sigma-Aldrich) (from a 100 mM stock in water) induced gene expression from the T71ac promoter. Cas mutants expressed from the pLtetO promoter were induced by anhydrotetracycline (aTc; Clontech) at a final concentration of 214 nM (from a 214 μM stock solution in 50% ethanol). Concurrently with expression from the pLtetO promoter, 0.2% glucose was added to reduce unintended background expression from the T71ac promoter. For new spacer harvesting experiments not involving oligonucleotide-derived spacers, cells were induced and grown overn...

Embodiment 2

[0089] Type I-E CRISPR-Cas systems accept synthetic spacers in vivo.

[0090] It was recently shown that overexpression of the Escherichia coli type I-E CRISPR-Cas proteins Cas1 and Cas2 is sufficient to drive acquisition of new spacers in a strain (BL21-AI) containing two genomic CRISPR arrays but lacking endogenous Cas proteins (see I. Yosef , M.G.Goren, U.Qimron, Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic acids research 40, 5569-5576 (2012); Epub online publication , July (10.1093 / nar / gks216)). The result is replicated (see Figure 1A ), and similarly a new spacer was found to be consistently integrated into the first position of Array I, which is directly adjacent to the leader, with a consistent size of 33 bases (see Figure 2A -B). Roughly equal numbers of these spacers were extracted from the cell's own genome and from the plasmids used to overexpress Cas1 and Cas2 (see Figure 1B ). Considering the overall DN...

Embodiment 3

[0095] PAM changes the efficiency and directionality of spacer acquisition.

[0096] Using data collected by our sequencing of millions of arrays after amplification, it was found that genomic and plasmid-derived protospacers were overall drawn in equal numbers from the forward and reverse strands, with the only apparent overall bias pointing towards genome duplication starting point (see Figure 3A ). Similarly, oligonucleotide-derived protospacers were also found to have equal proportions in the forward and reverse directions in the array (see Figure 3B ). When the context of the genomic and plasmid-derived protospacer was further examined, strong evidence for a PAM at the 5' end of the protospacer was found, consisting of two adenines from positions -2 and -1 of the spacer, and Strong preference for guanine as the first spacer base (see Figure 3C ). This is largely consistent with previous characterization of E. coli type I-E systems (see I. Yosef, D. Shitrit, M.G. G...

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Abstract

This invention provides methods of altering a cell including providing the cell with a nucleic acid sequence encoding a Cas1 protein and / or a Cas2 protein of a CRISPR adaptation system, providing thecell with a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence, wherein the cell expresses the Cas1 protein and / or the Cas2 protein and wherein the CRISPRarray nucleic acid sequence is within genomic DNA of the cell or on a plasmid. Also provided are methods and systems for nucleic acid storage and in vivo molecular recordings of events into a cell.

Description

[0001] relevant application data [0002] This application claims priority to U.S. Provisional Application No. 62 / 296,812, filed February 18, 2016, and U.S. Provisional Application No. 62 / 395,738, filed September 16, 2016, the entire contents of which are incorporated herein by reference for all purposes. [0003] Statement of Government Interests [0004] This invention was made with government support under Grant Nos. AG000222, MH103910 and NS045523 awarded by the National Institutes of Health. The government has certain rights in this invention. Background technique [0005] DNA has unparalleled advantages in encoding, preserving and disseminating information (GM Church, Y. Gao, S. Kosuri, Next-generation digital information storage in DNA), Science 337, 1628 (2012) ; Epub online, Sept. 28 (10.1126 / science.1226355)). The sharp drop in the cost of DNA sequencing now makes it practical to read out this information on a large scale (J. Shendure, H. Ji, Next-generation DNA s...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N9/16C12Q1/6869C07H21/04
CPCC12N15/10C12N15/102G06N3/123C12N2310/20C12N9/22C12N15/113C12N2310/122
Inventor G·M·丘奇S·L·希普曼J·D·麦克里斯J·M·尼瓦拉
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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