WO2020228844A2 - Procédé de test de l'activité d'un réactif générant une cassure double brin - Google Patents

Procédé de test de l'activité d'un réactif générant une cassure double brin Download PDF

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WO2020228844A2
WO2020228844A2 PCT/CN2020/098360 CN2020098360W WO2020228844A2 WO 2020228844 A2 WO2020228844 A2 WO 2020228844A2 CN 2020098360 W CN2020098360 W CN 2020098360W WO 2020228844 A2 WO2020228844 A2 WO 2020228844A2
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double
reagent
target
strand break
nucleic acid
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胡家志
尹健行
刘孟竺
刘阳
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北京大学
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Definitions

  • the present invention provides a method for simultaneously detecting the editing efficiency and specificity of double-strand break producing reagents, especially engineered nucleases.
  • TALEN transcription activator-like effector nuclease
  • CRISPR/Cas clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins
  • CRISPR/Cas derived from the immune system of bacteria and archaea, can use target site-specific RNA to guide Cas protein to modify the target site sequence. Due to its high efficiency and simplicity, it has caused widespread Attention has become a new and efficient genome editing tool.
  • targeted high-throughput sequencing is widely used to evaluate indels in genome fragments amplified by PCR (Mali et al., 2013 ), these data can be used to roughly estimate the cleavage efficiency of nucleases, but these methods cannot estimate the specificity of nucleases; while LAM-HTGTS uses whole-genome translocations of target gene sites as bait to identify off-target sites ( Frock et al., 2015) has the ability to detect its editing specificity.
  • this method first performs 80 cycles of linear amplification to generate multiple copies of the original DNA fragment, which makes it difficult to distinguish the PCR amplification product from the original template.
  • restriction enzyme blockade during library preparation leads to underestimation of uncut or completely repaired target fragments and small inserts, and therefore cannot quantify the DSB repair products around the target site (Hu et al., 2016), so this method cannot be effective Quantitative analysis of cutting efficiency.
  • the present invention provides a method that can simultaneously quantitatively analyze the editing efficiency of genome editing tools (such as engineered nucleases) (that is, the efficiency of cutting on the target target to generate double-strand breaks) and off-target sites.
  • genome editing tools such as engineered nucleases
  • This method is also the first method known in the art that can achieve the above objectives at the same time.
  • this method is also called PEM-seq method, which can be widely used for genome editing evaluation. in particular,
  • a method for detecting the activity of a double-strand break (DSB) generating reagent that can generate a double-strand break at a target location in the genome including:
  • step (2) Using the processed genomic nucleic acid in step (1) as a template, using an extension primer complementary to the sequence flanking the target position to perform an extension reaction to obtain an extension product whose sequence extends beyond the target position;
  • the bridge joint includes one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) lengths of A random nucleic acid segment of n nucleotides, where n is 1-30 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; so that each extension product obtains the random nucleic acid region by connecting the bridging linker Unique identifer assigned by the paragraph; and
  • step (3) Perform high-throughput sequencing on the ligation product of the extension product obtained in step (3) and the bridging linker to identify whether a double-strand break event occurs at a target location and/or a double-strand break event occurs at a non-target location.
  • the reagent includes a nuclease
  • the reagent is an engineered nuclease, such as zinc finger nuclease (ZFN), TALEN or CRISPR-CAS.
  • ZFN zinc finger nuclease
  • TALEN TALEN
  • CRISPR-CAS CRISPR-CAS
  • the sample is a eukaryotic cell, such as an animal cell (preferably a mammalian cell) or a plant cell, or a prokaryotic cell.
  • a eukaryotic cell such as an animal cell (preferably a mammalian cell) or a plant cell, or a prokaryotic cell.
  • step (2) before the extension reaction occurs, the extension primer and the sample genome are subjected to one or more denaturation-annealing cycles, preferably 1 to 20 times, for example, 1 time, 2 times , 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 Times or 20 times.
  • one or more denaturation-annealing cycles preferably 1 to 20 times, for example, 1 time, 2 times , 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 Times or 20 times.
  • the extension primer has an affinity tag, for example, an affinity tag, such as biotin, is attached to the 5'end of the extension primer.
  • step (1) after step (1), it further includes a step of fragmenting the genome, such as sonication or endonuclease digestion.
  • the binding site of the extension primer and the genomic nucleic acid is located within 2kb upstream or downstream of the double-strand break of the target position, such as 1kb, 900bp, 800bp, 700bp, 600bp Within 500 bp, 400 bp, 300 bp, 200 bp or 100 bp, it is also preferred that the extension primer is repeatedly annealed to the genome sequence.
  • the extension product in step (2) after obtaining the extension product in step (2), it further includes a step of separating the extension product; preferably, the separation using the affinity tag of the extension primer is carried out by affinity purification, more preferably The affinity purification is based on the binding between biotin and avidin or streptavidin. More preferably, the avidin or streptavidin is attached to a solid substrate, such as beads (bead ).
  • the activity refers to the cutting efficiency and/or specificity of the reagent; or, after the sequencing, the step of analyzing the cutting efficiency and/or specificity of the reagent according to the sequencing result .
  • a method for screening genomic double-strand break sites which includes
  • the reagent for producing double-strand breaks includes engineered nuclease, such as zinc finger nuclease (ZFN), TALEN or CRISPR-CAS9.
  • ZFN zinc finger nuclease
  • TALEN TALEN
  • CRISPR-CAS9 engineered nuclease
  • a method for screening engineered nucleases which includes:
  • the engineered nuclease is a Cas nuclease, more preferably the Cas nuclease is selected from Cas9, Cas12a, Cas12b, Cas13a, Cas14 and variants thereof.
  • a method for identifying a double-strand break generation reagent inhibitor which includes:
  • the reagent for producing double-strand breaks includes engineered nuclease, such as zinc finger nuclease (ZFN), TALEN or CRISPR-CAS9.
  • ZFN zinc finger nuclease
  • TALEN TALEN
  • CRISPR-CAS9 engineered nuclease
  • a method for identifying a double-strand break producing agent enhancer which includes:
  • the reagent for generating double-strand breaks includes engineered nuclease, such as zinc finger nuclease (ZFN), TALEN or CRISPR-CAS.
  • ZFN zinc finger nuclease
  • TALEN TALEN
  • CRISPR-CAS CRISPR-CAS
  • kits for use in the method described in any of the foregoing aspects is disclosed.
  • the kit includes extension primers complementary to the sequence flanking the target position, and a library of bridging linkers comprising the random nucleic acid sequence segment, wherein each bridging linker contained in the library
  • Each of the random nucleic acid sequence segments of has a unique sequence, so that each extension product obtains a unique identifer given by the random nucleic acid segment by connecting the bridging joint;
  • the kit further includes a reagent capable of generating a double-strand break (DSB) at the target site.
  • a reagent capable of generating a double-strand break (DSB) at the target site.
  • the reagent is an engineered nuclease, such as zinc finger nuclease (ZFN), TALEN or CRISPR-CAS.
  • ZFN zinc finger nuclease
  • TALEN TALEN
  • CRISPR-CAS CRISPR-CAS
  • a Cas9 protein which has the activity of generating double-strand breaks at the target position of the genome, and its amino acid sequence is at least 80%, 85% and 85% to the sequence shown in SEQ ID NO:1. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, and selected from lysine (K) at position 848, 1003 There are mutations in one or more positions of lysine (K), arginine (R) at position 1060, and aspartic acid (D) at position 1135.
  • nucleic acid sequence encoding the protein sequence described in SEQ ID NO: 1 is as follows:
  • the mutations are specifically K848A, K1003A, R1060A, and D1135E.
  • sequence of the Cas9 protein is shown in SEQ ID NO: 3.
  • a nucleic acid sequence encoding the Cas9 protein of the eighth aspect an expression vector for expressing the nucleic acid sequence, and a cell containing the nucleic acid sequence and the expression vector are disclosed
  • eukaryotic cells specifically plant cells, animal cells (preferably mammalian cells), or prokaryotic cells.
  • nucleic acid sequence encoding the Cas9 protein of the seventh aspect is preferably the nucleic acid sequence shown in SEQ ID NO: 4:
  • the application of the aforementioned Cas9 protein, nucleic acid sequence, expression vector or cell in genome targeted modification is provided.
  • the method provided by the present invention can be used to evaluate the editing ability of engineered nucleases including CRISPR/Cas9, which is significantly better than existing methods in the prior art.
  • the main reasons are: first, the use of primer extension and molecular beacon (RMB) eliminates the amplification bias in the PCR amplification process used in other methods (such as T7EI, RFLP and targeted sequencing); secondly, PEM- seq can comprehensively detect the insertion and deletion of genome-size fragments generated after gene editing, as well as genome-wide translocations. These are the events that occur in CRISPR/Cas9 editing.
  • RMB primer extension and molecular beacon
  • T7EI T7EI
  • RFLP target Toward sequencing
  • target Toward sequencing can only be used to detect small indels, or can only be used to analyze off-target sites (such as LAM-HTGTS). Therefore, the method of the present invention has immeasurable value in the evaluation, detection, analysis, and screening of genome editing tools.
  • Figure 1 shows the results of using PEM-Seq to detect off-target hot spots of CRISPR/Cas9.
  • A Overview of PEM-seq.
  • a primer extension reaction using biotinylated primers is used to obtain a single copy template, and then a bridge adapter is connected and DNA amplification is performed.
  • the solid line represents the bait area, and the dashed line represents the captured genome area.
  • N represents the random nucleic acid sequence in the bridging linker, which can also be called a molecular beacon (RMB).
  • the arrow shows the location and direction of the primer.
  • B SpCas9: Circos plot of the RAG1A library.
  • a total of three biological repeats are displayed from the outside to the inside, and the displayed translocation connections are 19,494, 16,005 and 18,078 respectively.
  • the whole genome translocations divided into 5-Mb regions are plotted on a logarithmic scale.
  • the chromosomes are displayed in a clockwise direction from centromere to telomere.
  • the black arrow indicates the SpCas9:RAG1A cleavage site.
  • the lines in the inner ring connect the target point to the off-target hot spot.
  • C An enlarged view of SpCas9:RAG1A translocation junction (bin is 2Mb) on chromosome 7. The black arrows indicate the identified off-target hot spots.
  • Cen represents the centromere
  • p/q represents the chromosome arm.
  • the Pearson correlation coefficient between repetitions 1 and 2 was 0.99, between repetitions 1 and 3 was 0.99, and between repetitions 2 and 3 was 0.98.
  • D Scatter plots of SpCas9:RAG1A off-target hot spots in 293T, HCT116, K562 and U2OS cells. The y-axis shows the frequency of each off-target hot spot in every 100,000 editing events (including indels and translocations). Asterisks indicate off-target hotspots detected by PEM-seq but not detected by LAM-HTGTS.
  • FIG. 1 Venn diagrams are used to show the overlapping relationship between SpCas9:RAG1A off-target hot spots in 293T, HCT116, K562 and U2OS cells. The legend is the same as the figure (D).
  • (F) In vitro SpCas9 digestion for RAG1A off-target hot spots. Incubate the indicated amplified fragments with purified SpCas9 for 20 hours. "On” indicates the target site of RAG1A. "NC”, does not carry the SpCas9:RAG1A target site, but can be targeted by SpCas9:MYC1, as a negative control.
  • the inverted triangle arrows indicate uncut segments, while the horizontal triangle arrows indicate larger cut segments.
  • FIG. 2 shows the results of using PEM-seq to detect the editing activity of CRISPR/Cas9.
  • A Cas9 induced double-strand break (DSB).
  • Germline represents uncut or perfect rejoining; indels (insertions) come from incorrect rejoining; translocation involves the second DSB.
  • B SpCas9: percentage of Germline, indels and translocations of RAG1A. Mean ⁇ SD.
  • C SpCas9: RAG1A indel frequency detected by PEM-seq, RFLP, T7EI assay and single cell RFLP. The average value is indicated by a black line. DNA from different repetitions is marked with different colors.
  • the small box represents the Cas9 target.
  • the tailless arrow indicates the direction of the translocation.
  • Arrows with dotted tails indicate the position and direction of primers used for PEM-seq.
  • the dark dashed line indicates the Cas9 cleavage site, and the light dashed line indicates the primer position.
  • the figure also shows the number of connections in each region;
  • SpCas9 the connection frequency of 5-50 kb downstream of the RAG1A cleavage site (bin is 1 kb).
  • the dashed line indicates the boundary of the 5-50kb region.
  • Arrows with dotted tails indicate primers used for PEM-seq.
  • the number of connections in each area is displayed.
  • Figure 3 shows the difference in editing ability between different targets in the surrounding area of RAG1A.
  • the figure above is a schematic diagram of the target sites of RAG1A, RAG1B, RAG1C and RAG1G.
  • the arrow with a dotted tail indicates the biotinylated primer used for PEM-seq.
  • the light-colored long box indicates the gRNA target site, and the dark block in the box indicates the Cas9 cleavage site.
  • the figure below shows the respective components of the germline, indels and translocations produced by Cas9 treatment in HEK293T cells. Mean ⁇ SD. For details, see Tables S1 and S3.
  • Figure 4(A) is a schematic diagram of the SpCas9 domain and corresponding point mutations of SpCas9 variants.
  • (B) Editing efficiency of each SpCas9 variant targeting RAG1A in HEK293T cells detected by PEM-seq. The upper part lists the target site sequence, and the underlined bases represent the PAM sequence. Error bar, mean ⁇ SD; two-tailed t-test, *, p ⁇ 0.05;
  • C For specific SpCas9 variants targeting RAG1A in HEK293T cells, the frequency of total translocation junctions in the 1kb region around off-target hot spots . Above the bar shows the total number of off-target hot spots identified for each Cas9 variant.
  • FIG. 1 shows the off-target hot spots of each SpCas9 variant when targeting the EMX1 target site.
  • the y-axis shows the frequency of each hot spot per 100,000 editing events (indels+translocation).
  • HJ When targeting MYC target site 1 (locus 1) in HEK293T cells, the editing efficiency and off-target hot spots of each SpCas9 variant;
  • KM shows a statistical comparison of the off-target hot spots between eSpCas9 and FeSpCas9 at the three gene targets result.
  • Figure 5 shows the application of PEM-seq in evaluating the editing efficiency and specificity of SpCas9, SaCas9 and AsCas12a.
  • A shows the target DNA sequence and PAM sequence of SpCas9, SaCas9 and AsCas12a. The letters indicate the PAM sequences of different Cas9 enzymes, where blue represents SpCas9, green plus blue represents SaCas9, and red represents AsCas12a.
  • B Editing efficiency of SpCas9 (Sp), SaCas9 (Sa) and AsCas12a (As) against selected targets in HEK293T cells. Error bars, mean ⁇ SD.
  • Figure 6(A) shows the editing efficiency of SpCas9:RAG1A in HEK293T cells using the PEM-seq method when the mass ratio of Cas9 and AcrIIA4 is shown in the figure. Error bars, mean ⁇ SD. Two-tailed t test, **, p ⁇ 0.01.
  • (B) shows the frequency of translocation connections in the 1-kb region near the SpCas9:RAG1A off-target hot spot in HEK293T cells when the mass ratio of Cas9 to AcrIIA4 is shown in the figure. The total number of off-target hot spots is shown above the bar. Error bars, mean ⁇ SD. Two-tailed t test, *, p ⁇ 0.05; **p ⁇ 0.01.
  • (C) shows the composition ratio of indels and off-target ligation in the SpCas9:RAG1A sequencing result library with Cas9 and AcrIIA4 in the ratio shown in the figure.
  • the total number of connections from the three sequencing library is shown above the bar.
  • (D-G) respectively show the editing efficiency and off-target hot spots of SpCas9 against other target sites.
  • the mass ratio of Cas9 to AcrIIA4 is 1:1.
  • the legend is the same as (A-C).
  • target position refers to the position in the genome that is selected or targeted for the occurrence of a double-strand break, which can mean the actual specific nucleotide position of the double-strand break, or it can mean that it is selected as the target
  • the target sequence includes a nucleic acid sequence of 1 or more nucleotides.
  • double-strand break event refers to the occurrence of a DNA double-strand break (DSB) at a specific location (or a target location) in the sample genome.
  • DNA double-strand break events are events that occur in the body (in cells), and cells have a DNA repair mechanism. Therefore, after a DNA double-strand break occurs, the cell will repair the double-strand break and may produce various repair results: If a perfect repair occurs, the repaired target position sequence is the same as before the double-strand break; if it is not perfect, the repaired target position sequence may be compared with the original sequence, and partial nucleotide insertion or insertion may occur. Deletions (indels), or exchange with nucleic acid fragments elsewhere in the genome to produce translocations. Therefore, in a broader sense, "double-strand break event” also includes the DNA repair process that occurs after a DNA double-strand break occurs in the genome.
  • double-strand break-generating reagent refers to a reagent that cuts a specific position in the genome to generate a DNA double-strand break, and includes a protein (such as an enzyme) or a nucleic acid, or a combination of more than one protein or nucleic acid.
  • the cleavage is preferably cleavage at a specified position in the genome, that is, targeted cleavage.
  • the reagent When targeted cleavage is performed, the reagent preferably includes a targeting reagent and a cleavage reagent, or the reagent includes a targeting moiety and a cleavage moiety, such as a DNA fragment that can bind to a target location in the genome, a targeting moiety such as a protein (such as The domains of enzymes, such as nucleases, which bind to DNA, the targeting agent or the targeting moiety can guide the cleavage agent or the cleavage moiety to produce a double-strand break at the target location.
  • a targeting reagent includes a targeting moiety and a cleavage moiety, such as a DNA fragment that can bind to a target location in the genome, a targeting moiety such as a protein (such as The domains of enzymes, such as nucleases, which bind to DNA, the targeting agent or the targeting moiety can guide the cleavage agent or the cleavage moiety to produce a double-strand
  • the double-strand break producing reagent is a genome editing tool.
  • the double-strand break producing reagent is an engineered nuclease
  • the engineered nuclease refers to an engineered nuclease, for example, the amino acid sequence of the nuclease is improved or optimized, or its auxiliary sequence (for example, auxiliary guide After the DNA or RNA sequence that the enzyme cuts) is optimized, the nuclease can achieve specific cutting or targeted cutting according to requirements.
  • the engineered nuclease is not only the nuclease itself, Other auxiliary sequences are also included. Examples of such engineered nucleases are zinc finger nuclease (ZFN), TALEN or CRISPR-CAS.
  • ZFN is a chimeric protein molecule that can promote double-strand break (DSB) at a target gene site in a host cell.
  • the ZFN may include a DNA binding domain and a DNA cleavage domain, wherein the DNA binding domain includes at least one zinc finger and is connected to the DNA cleavage domain.
  • the zinc finger DNA binding domain is at the N-terminus of the chimeric protein molecule and the DM cleavage domain is at the C-terminus of the molecule.
  • the ZFN DNA cleavage domain can be derived from a non-specific DNA cleavage domain, such as the DNA-cleavage domain of a class II restriction endonuclease.
  • the DNA cleavage domain is derived from Fok I nuclease.
  • TALEN transcription activator-like effector nuclease
  • TALE transcription activator-like effector
  • TALEs are composed of 12 or more tandem "protein modules" that specifically recognize DNA and N-terminal and C-terminal sequences on both sides. Most of the amino acid sequences of this series of modules are repeated, and the difference is basically in the 12 and 13 positions of the repeated sequence. These repeated amino acid sequences are called repeated variable sequences (RVDs). Among them, the 12th and 13th residues are the key sites for targeted recognition and are called di-residues. Different from the specific triplet bases that each zinc finger protein recognizes, each RVDs on TALEs can only recognize one base, and there is no obvious front-to-back nucleic acid dependency, so TALEs can be designed to recognize and bind all target DNA sequence.
  • RVDs repeated variable sequences
  • CRISPR-Cas is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight invading viruses and foreign DNA.
  • the CRISPR-Cas9 system integrates fragments of invading phage and plasmid DNA into CRISPR, and uses corresponding CRISPR RNAs (crRNAs) to guide the degradation of homologous sequences to provide immunity.
  • crRNA CRISPR-derived RNA
  • tracrRNA trans-activating RNA
  • CRISPR-associated protein CRISPR-associated protein, such as Cas9, etc.
  • sgRNA short guide RNA
  • extension primer refers to a primer that is complementary to a flanking sequence at a target location (or the location where a DSB break event occurs), and the flanking sequence includes upstream or downstream of the target location (or DSB break event), and the extension primer It is a single primer that binds to the upstream or downstream of the DSB site caused by the double-strand break generating reagent by annealing. After the extension reaction occurs, the extension product is obtained, and it is a single copy of the template, thus retaining the original quantitative information of the template DNA. The extension length of the extension product at least needs to exceed the target location (or the location where the DSB fracture occurs).
  • the mixture is subjected to 1 or more times (preferably 1 to 20 times, such as 1 time, 2 times, 3 times, 4 times, 5 times). Times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times or 20 times) Denaturation-Annealing The loop.
  • One cycle of genome denaturation and primer annealing to the target binding site is one cycle. On this basis, genome denaturation and primer annealing are the next cycle. The inventor found that repeated annealing treatment can greatly improve the binding efficiency of the primer and the template and achieve complete coverage of the template.
  • bridging linker refers to a random nucleic acid segment containing one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of n nucleotides in length (In this article, sometimes a random nucleic acid segment is also called a random molecular beacon (RMB) or molecular beacon, which has the same meaning).
  • RMB random molecular beacon
  • Different bridging joints pass through the random sequence in the random nucleic acid segment. The difference is different from each other.
  • n is 1-30 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.
  • each of the bridging linkers includes one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) shared nucleic acid regions in addition to random nucleic acid segments.
  • Segment, the length of the shared nucleic acid segment is m, and m is 1-30 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.
  • the shared nucleic acid segment and the random nucleic acid segment are arranged at random intervals.
  • both ends of the bridging linker are shared nucleic acid segments, and when the bridging linker is connected to the extension primer, the shared nucleic acid segment is located at the outer end (or distal end)
  • the nucleic acid segments can be bound by primers to facilitate subsequent sequencing.
  • the shared nucleic acid segment portion includes the same nucleotide sequence in each bridging linker molecule.
  • the bridging linker includes a random nucleic acid segment and two common nucleic acid segments on both sides.
  • a bridging linker is a double-stranded DNA fragment, which can be blunt ends on both sides, blunt ends on one side, and sticky ends on the other side, or both sides are sticky ends.
  • the double-stranded DNA fragment passes through two Single-stranded DNA is formed by annealing.
  • each extension product is connected to the bridging linker, a unique identifer given by the random nucleic acid segment is obtained, that is, the end of each extension product is connected to a bridging linker with a unique sequence (the uniqueness is determined by Given by random nucleic acid segments).
  • editing efficiency also referred to as “cleavage efficiency” in the present invention, refers to the efficiency of double-strand break-generating reagents at the target genomic site to generate double-strand breaks, that is, the template that is cleaved at the target site after cutting The ratio of the amount to the total amount of DNA template.
  • the editing efficiency reflects the ability of the reagent or gene editing tool to act on the target location.
  • the term "specificity" refers to the fact that the reagents that produce double-strand breaks sometimes inevitably cleave at other locations (ie off-target sites) in the genome other than the target site. Specificity is used to measure the target in all cleavage events. The proportion occupied by positional cutting, the higher the proportion, the better the specificity. It should be noted that in gene editing, the specificity is not only related to the tools used in gene editing, but also related to the location of the selected target. For example, when editing the same gene, choosing different targets may also result in different specificities.
  • 1.2 ⁇ AxyPrep Mag PCR Clean Up bead (Axygen, USA) was added to remove excess biotinylated primers.
  • the purified product was heated to 95°C for 5 minutes, and then quickly cooled on ice for 5 minutes to denature the DNA.
  • Dynabeads TM MyOne TM Sterptavidin C1 (Thermo Fisher) was used to enrich biotin-labeled extension products.
  • the extension products on Streptavidin C1 beads were washed twice with 400 ⁇ L 1 ⁇ B&W buffer (1M NaCl, 5mM Tris-HCl (pH 7.4) and 1mM EDTA (pH 8.0)), and then washed with 400 ⁇ L dH 2 O.
  • the DNA-bead complex was then resuspended in 42.4 ⁇ L dH 2 O.
  • T4DNA ligase (Thermo Fisher Scientific) is used to perform ligation reaction with the bridge adapter in 15% PEG8000 (Sigma) at room temperature.
  • the bridge adapter is formed by annealing two single-stranded DNAs. The sequence of each strand is as follows:
  • Bridge adapter-up /5phos/CCA CGC GTG CTC TAC ANN NNT NNN ANN NTN NNN AGA TCG GAA GAG CAC ACG TCT GAA CTC CAG T-NH 2 (C7) (SEQ ID NO: 25);
  • Bridge adapter-lower TGT AGA GCA CGC GTG GNN NNN N-NH 2 (C7) (SEQ ID NO: 26).
  • N is optional A, T, C or G.
  • connection reaction reaction system is as follows:
  • the ligation product was washed successively twice with 400 ⁇ L 1 ⁇ B & W buffer and 400 ⁇ L dH 2 O and then with 80 ⁇ L dH 2 O suspension.
  • the DNA-bead complex was directly subjected to nested PCR (Taq, Transgen Biotech, China) with I5 and I7 sequence primers on the bead, and amplified 16 cycles.
  • the PCR product was then recycled through size-selection beads (Axygen, USA), followed by PCR (Fastpfu, Transgen Biotech, China), and labeled Illumina P5 and P7 sequences. All the PEM-seq libraries constructed above were sequenced with 2 ⁇ 150bp Hiseq.
  • the targeting gRNA of SpCas9 uses the pX330 backbone (Addgene 42230), and the SpCas9 nickase uses the pX335 backbone (Addgene 42335). Insert the SpCas9 variants, SaCas9 and AsCpf1 into the pX330 backbone, the specific steps are as follows:
  • the cDNAs of SpCas9 variants were generated by mutation-overlap PCR, and were digested and ligated into the pX330 plasmid using AgeI/EcoRI. After digesting pX601 (Addgene 61591) with AgeI/EcoRI, SaCas9cDNA was purified, and then ligated into the pX330 plasmid that was also digested with AgeI/EcoRI.
  • the plasmid U6 promoter-SaCas9gRNA scaffold DNA from pX601 was inserted between the AflIII and XbaI sites of the pX330-SaCas9 plasmid.
  • AsCas12a (Cpf1) cDNA was amplified from SQT1659 (Addgene78743) and inserted between the AgeI and EcoRI sites of pX330;
  • Cas12a gRNA scaffold DNA was inserted into the pX330-AsCfp1 backbone that was double digested with BbsI and XbaI.
  • AcrIIA4 plasmid PJH376 was purchased from Addgene (Addgene 86842).
  • the 293T, U2OS and HCT116 cells were cultured in DMEM (Corning) medium supplemented with glutamine (Corning), 10% FBS, and penicillin/streptomycin (Corning) at 37°C and 5% carbon dioxide.
  • K562 cells were cultured in RPMI 1640 (Corning) containing glutamine, 15% FBS and penicillin/streptomycin (Corning) at 37°C and 5% carbon dioxide.
  • the HEK293T cell library was prepared by co-transfection with 7.2 ⁇ g nuclease plasmid and 1.8 ⁇ g pMAX-GFP with Ca-PO 4 .
  • U2OS library was co-transfected with 20 ⁇ g Cas9 plasmid and 5 ⁇ gGFP plasmid using PEI (Sigma). Also in a 10 cm culture dish, 20 ⁇ g Cas9 plasmid and 5 ⁇ g GFP plasmid were transfected into HCT116 cells using Lipofectamine 2000 (Invitrogen). In SF buffer (Lonza), 20 ⁇ g of pX330 and 5 ⁇ g of GFP were co-transformed into K562 cells using nucleofector 4D combined with the FF120 program.
  • the Hiseq reads are processed as follows.
  • Illumina adaptor sequences and end low-quality sequences (QC ⁇ 30) were removed using the cutadapt program (http://cutadapt.readthedocs.io/en/stable/); at the same time, reads with remaining sequences shorter than 25bp were removed.
  • the fastq-multx program https://github.com/brwnj/fastq-multx
  • S represents the nuclease-treated library
  • C represents the control library
  • the translocation junctions located within ⁇ 250kb of the break site were excluded for subsequent analysis.
  • the overlap between the decoy sequence (ie the sequence near the double-strand break at the target site of interest) and the capture sequence (ie the sequence near the double-strand break at the off-target site) is considered microscopic homology (deletion); between the decoy and the capture sequence
  • the gap is considered to be inserted. Then calculate the percentage of 0 to 10 nucleotide insertions and deletions.
  • the RAG1A locus was amplified by Fastpfu (Transgen Biotech, China) PCR.
  • the amplified product was recovered by 1.2 ⁇ AxyPrep Mag PCR Clean-Up beads (Axygen, US), then cut with StyI (NEB) for 1 hour, and then subjected to agarose gel electrophoresis.
  • Quantify the band intensity by Image J (version 1.51J8), use the following formula to measure indels:
  • I C is the sum of the strength of the two strips that are cut, and I U is the strength of the uncut strip.
  • the overall process refers to the previously described method (Brinkman et al., 2014).
  • Design primers for the target site of RAG1A Genomic DNA was extracted from SpCas9RAG1A transfected cells, and 50ng of genomic DNA was used as a template, and conventional PCR procedures were used to amplify for 30 cycles. Prepare gel-purified PCR products for Sanger sequencing.
  • the result file (.ab1) is analyzed by the tool provided by the https://tide.deskgen.com/ website.
  • Chromatin translocations and indels caused by non-specific cleavage of CRISPR/Cas9 may threaten the stability of the cell genome after treatment. Therefore, it is necessary to effectively evaluate the specificity and efficiency of CRISPR/Cas9.
  • PEM-seq Use primer-extension-mediated sequencing (PEM-seq) to extend from the primer binding site, then connect a bridging adapter containing a random molecular beacon (RMB) to the end, and then construct the library And sequencing, and then get the DSB information through bioinformatics analysis.
  • RMB random molecular beacon
  • the method of the present invention is also called PEM-seq method.
  • the present invention uses a primer extension method to generate a single copy product of the original template. After these fragments are separated, each fragment is connected to a unique sequence and length (in the subsequent implementation) In the example, the length of the random nucleic acid sequence is 14bp), and the adaptor of the random nucleic acid sequence (also called molecular beacon (RMB)) can specifically label each fragment ( Figure 1A), thereby avoiding the preference of amplification and thus The original proportions of the perfect match and the mutation fragments are retained, thereby retaining the original quantitative information.
  • RMB molecular beacon
  • Chromatin heterotopia is the connection of two independent double-strand breaks (DSBs). Setting a site-specific primer at a known induced DSB site can help identify another unknown DSB. Therefore, the PEM-seq method can accurately detect the location of chromatin translocation and quantify chromatin heterotopia. In addition, because PEM-seq retains the original quantitative information and does not introduce other processing (such as restriction enzyme digestion) that will nearby DSB structures, PEM-seq can also quantitatively detect indels and other DSB results.
  • Example 2 PEM-seq can detect off-target hot spots of CRISPR/Cas9 very sensitively
  • PEM-seq identified a total of 53 off-target sites, including 24 new sites that were not identified by LAM-HTGTS, and lost 4 weak sites identified by LAM-HTGTS ( Figure 1B-D and Table S2). ). In order to verify the authenticity of these off-target sites, 8 of them were amplified next, including 4 new sites and 4 common sites, as well as the correct cleavage target sites in the RAG1A gene (on-target sites). ) And use them for in vitro CRISPR/Cas9 processing.
  • LAM-HTGTS can only be used to assess off-target sites, and cannot detect other events generated after cleavage, such as indels.
  • PEM-seq can be used for quantitative detection All editing events to make a better assessment of nucleic acid editing events.
  • Example 3 PEM-seq can quantitatively analyze the editing ability of CRISPR/Cas9
  • Chromatin heterotopia is the connection of two independent double-strand breaks (DSBs), for example, between the target DSB and the DSB that Cas9 cleaves off-target, or the basal level DSB that naturally exists in the genome; indels are caused by Cas9
  • DSBs double-strand breaks
  • Figure 2A The so-called "germline” refers to the target segment that has not been cut or is completely repaired after being cut. In the case of complete repair, the completely repaired segment cannot be distinguished from the uncut situation. Therefore, the uncut segment and the completely repaired segment are usually The fragments are considered to be no cutting event.
  • SpCas9:RAG1A was transfected into HEK293T cells and incubated for 48 hours.
  • the detected translocation and indel levels were 2.7% and 35.7%, respectively, and the total editing efficiency was about 38.4% ( Figure 2B and Table S1).
  • the methods commonly used in the prior art to evaluate the efficiency of indels RFLP, T7EI, and single cell-RFLP were used to analyze the CRISPR/Cas9 cleavage at the RAG1A site, and the results showed the ratio of indels detected by these methods It is 27-40% (Figure 2C). It can be seen that the ability of PEM-seq to detect indels is similar to existing methods in the prior art.
  • PEM-seq can reliably quantitatively detect indels at the target site, so as to evaluate the editing efficiency of CRISPR/Cas9.
  • PEM-seq can also detect off-target sites by detecting translocations, which cannot be achieved by the above three methods (RFLP, T7EI and single cell-RFLP).
  • eSpCas9 was placed behind the same promoter as wild-type SpCas9 and designed to target the RAG1A site of HEK293T cells. As expected, eSpCas9 only produced 7 off-target sites, and there was no significant loss in editing efficiency (Figure 4B-D, Table S4).
  • Example 5 PEM-seq is used to evaluate the editing efficiency and specificity of different CRISPR-Cas nucleases against the same target site
  • this example selects three Cas nucleases known in the art: SpCas9 , SaCas9 and AsCas12a. At the same time, 4 target sites near two genes were selected in the genome (2 are near the c-MYC gene, and the other 2 target sites are near the DNMT1 gene). The PEM-seq method was used to The Cas9 nuclease was evaluated ( Figure 5A). In the experiment, in order to ensure that the above-mentioned nuclease has a similar expression level in the cell, the same plasmid backbone was used and the three Cas genes were all placed behind the chicken ⁇ -actin promoter.
  • This example uses the PEM-seq method to test the ability of the widely used SpCas9 inhibitor AcrIIA4 to block SpCas9:RAG1A in HEK293T cells.
  • the plasmids expressing SpCas9 AcrIIA4 were co-transfected, and the mass ratio was from 3:1 to 1:1, and then to 1:3.
  • the results showed that when AcrIIA4 was co-transfected, the editing efficiency of SpCas9 was significantly reduced. Among them, when the transfection ratio is 3:1, the editing efficiency of SpCas9 is reduced by 11 times.
  • the ratio of AcrIIA4 is increased to 1:1 and 1:3, the SpCas9 activity is further inhibited ( Figure 6A).

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Abstract

La présente invention concerne un procédé de test de l'activité d'un réactif générant une cassure double brin. Au moyen de la conception d'une amorce étendue et d'une balise moléculaire à séquence aléatoire, l'invention peut permettre d'obtenir un double test de l'efficacité d'édition et de la spécificité du réactif.
PCT/CN2020/098360 2019-03-15 2020-06-28 Procédé de test de l'activité d'un réactif générant une cassure double brin WO2020228844A2 (fr)

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