CN110343751A - A method of detection CRISPR-Cas9 undershooting-effect - Google Patents
A method of detection CRISPR-Cas9 undershooting-effect Download PDFInfo
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Abstract
The invention discloses a kind of methods for detecting CRISPR-Cas9 undershooting-effect, belong to gene engineering technology field, step includes: a. the site of missing the target using software prediction sgRNA, and obtains PCR amplification primer；B. cdna sample is chosen, selected cdna sample includes gene editing sample and do not carry out gene editing sample；C.PCR expands the site of missing the target predicted；D. high-flux sequence is carried out to PCR product；E. interpretation of result, sequence alignment；The present invention is according to specified comparison rules, sample after the gene editing is compared with the check sample for not carrying out gene editing, and it is not only compared with the information in data library, it does not need to obtain filial generation or parent's sample gene information yet, realize that the personalized possibility looked in cellular genome is missed the target site, the accuracy and sensitivity for improving detection of missing the target, to improve the safety of gene editing.
Description
Technical field
The present invention relates to gene engineering technology field more particularly to a kind of methods for detecting CRISPR-Cas9 undershooting-effect.
Background technique
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system
It is to research and develop a kind of successfully gene editing system in recent years, is a kind of bacterium acquired immune system.Due to easy to operate, fast
The advantages that efficient, the CRISPR/Cas9 system from streptococcus pyogenes are widely used in genetic engineering.CRISPR/Cas9
System includes Cas9, crRNA and tracrRNA, during CRISPR/Cas9 system functions, Cas9 albumen,
CrRNA and tracrRNA is combined form compound first, identifies and combine the complementary series of crRNA, the HNH of subsequent Cas9 albumen
Active site shears the complementary dna chain of crRNA, and Ruvc active site shears incomplementarity chain, is broken DNA double chain, realizes to DNA
Fixed point editor.
However, i.e. cutting and targeting sequence is similar studies have shown that CRISPR/Cas9 system is there are serious undershooting-effect
As non-target spot regional DNA sequence.The generation of undershooting-effect may destroy the function of non-targeted gene, for example, proto-oncogene and
Tumor suppressor gene, even a wide range of genetic recombination.This disadvantage seriously limits CRISPR/Cas9 system in biomedicine
The utilization in field.
Currently, the method for detection of missing the target mainly has T7EN1 enzyme cutting method, IDLV method, GUIDE-seq technology and Digenome-
Seq technology etc., these methods respectively have advantage and disadvantage:
The operation of T7EN1 enzyme cutting method is most simple, time-consuming most short, but sensitivity is too low, can only detect the higher site of off-target rate；
GUIDE-seq technology is mainly that breaking for CRISPR/Cas induction is marked using a kind of short double chain oligonucleotide label
It splits and (in target and misses the target), high-flux sequence, this method entire detection process time then are carried out to the gene region where label
It is longer, complex steps, and cannot be guaranteed that all broken sites are all labeled；
And Digenome-seq technology is only used in unicellular, and equally exists that accuracy is not high enough to be lacked
Point.
Therefore, develop that a kind of accuracy is high, the detection method of missing the target of high sensitivity is still very necessary.
Summary of the invention
The object of the invention is that a kind of method of completely new detection CRISPR-Cas9 undershooting-effect is provided, to solve
The above problem.
To achieve the goals above, the technical solution adopted by the present invention is that such: a kind of detection CRISPR-Cas9 misses the target
The method of effect comprising following steps:
A. it predicts the site of missing the target of sgRNA, and obtains PCR amplification primer；
B. cdna sample is chosen, the cdna sample includes gene editing sample and do not carry out gene editing sample；
C. PCR amplification is carried out to the site of missing the target that step (a) predicts；
D. high-flux sequence is carried out to the resulting PCR product of step (c)；
E. interpretation of result, sequence alignment.
As a preferred technical scheme: in step (a), the method in the site of missing the target of the prediction sgRNA is to pass through software
Or website is predicted.
As further preferred technical solution: the software or website are selected from seqmap, CRISPOR, CRISPR
Appointing in finder, CRISPR Design, sgRNAcas9, CRISPRdirect, COSMID, Off-Spotter, E-CRISP
One.
As further preferred technical solution: being screening scoring highest by the method that the software or website are predicted
At least 3 potential sites of missing the target.Selection for the item number of potential point of missing the target, item number is more, the accuracy of testing result
It is higher；But item number is more, testing cost is also higher；The present invention further preferred 6, accuracy and cost can be taken into account.
As a preferred technical scheme: in step (c), PCR reaction system are as follows: Transtar taq 0.5 μ l, 10 ×
Buffer 2.5 μ l, dNTP 2 μ l, 1 μ l of forward primer (10 μM), 1 μ l of reverse primer (10 μM), 1 μ l of DNA, nuclease-free water
Add to 25 μ l；Amplification condition: 95 DEG C of 5min, 95 DEG C of 30s, 68 DEG C of 30s (- 1 DEG C/circulation), 72 DEG C of 30s, 10 circulations, 95 DEG C
30s, 58 DEG C of 30s, 72 DEG C of 30s, 25 circulations, 72 DEG C of 5min.
As a preferred technical scheme: in step (d), high-flux sequence strategy includes: purified genomic dna, interrupts base
Because of a group DNA, end is repaired, 3 ' ends plus A tail, two end adapter of join index, validate library and cluster,
Sequencing.
As a preferred technical scheme: in step (e), the method for interpretation of result are as follows: pcr amplification product sequence passes through NCBI
Online primer blast is obtained, and refers to genome as customized；Software CASAVA is utilized to sequencing result raw image data
The identification of image base is carried out, preliminary quality analysis obtains the initial data of sequencing sample；Optimize original number using Cutadapt
According to；The data after optimization are merged using Pandaseq；Sequence after merging is made with the customized genome that refers to
It is compared with BWA software；Reuse samtools detection SNP/InDel.
As further preferred technical solution: result carries out preliminary screening, and filter condition is substrate quality value > 20, covers
Lid depth > 4 map mass value > 40.
Compared with the prior art, the advantages of the present invention are as follows: the present invention to choose the sample after carrying out gene editing and not
The check sample of gene editing is carried out, the potential site of missing the target of software prediction gene editing is then passed through, and obtains pcr amplification primer
Then object carries out high-flux sequence, last according to specified comparison rules, does not carry out the sample after the gene editing and base
Because the check sample of editor is compared, and be not only compared with the information in data library, do not need yet obtain filial generation or
Parent's sample gene information realizes that the personalized possibility looked in cellular genome is missed the target site, and the standard for detection of missing the target is improved
Exactness and sensitivity, to improve the safety of gene editing.
Detailed description of the invention
Fig. 1 is the undershooting-effect result figure of OT7 in the embodiment of the present invention 2；
Fig. 2 is the undershooting-effect result figure of OT13 in the embodiment of the present invention 2；
Fig. 3 is the undershooting-effect result figure of OT16 in the embodiment of the present invention 3；
Fig. 4 is the restriction enzyme digestion and electrophoresis result figure of comparative example.
In figure: site of missing the target is predicted in the expression of underscore part, and italic is the site PAM, and runic is mutational site, and "-" indicates
Base deletion.
Specific embodiment
It hereinafter, will be referring to embodiment the present invention is described in detail.However embodiments of the invention are herein only
It describes for purposes of illustration, and interest field of the invention is not only restricted to these embodiments.
Embodiment 1:
People's PD1 gene C RISPR/Cas9 gene editing sgRNA1 misses the target site primer:
Step 1: miss the target site estimation and the synthesis of PCR amplification primer.Predict that PD1 gene used is compiled using software seqmap
Collect the potential site of missing the target of sgRNA1.Specifically, by the site of missing the target of software prediction sgRNA1, forecasting software (website) includes
Seqmap, CRISPOR, CRISPR finder, CRISPR Design, sgRNAcas9, CRISPRdirect, COSMID, Off-
Spotter, E-CRISP etc., but not limited to this.6 potential sites of missing the target of screening scoring highest (possibility of missing the target highest) take off
Target site information is as shown in table 1.And leading-out needle is to the PCR amplification primer (table 2) in these sites of missing the target；
1 people's PD1 gene C RISPR/Cas9 gene editing sgRNA1 of table miss the target site screening
Underscore base indicates mispairing in table 1
Table 2 misses the target site PCR primer information
Step 2: the selection and extraction of genome sample.During the present invention is implemented, selected cdna sample includes compiling by gene
Volume after sample and be without the purpose of the sample of gene editing, in subsequent step, screen editing sample existing for InDel
When site, in addition to being compared with information in database, is also compared with without the sample of gene editing, do not need to obtain son
Generation or parental gene information realize that the personalized possibility looked in cellular genome is missed the target site；Using the genome of OMEGA
Extracts kit extracts No. 1 respectively, and No. 2, No. 3 patients do not edit the PBMC genome after peripheral blood PBMC and gene editing, base
Because edit methods referring to number of patent application are 2014100774746, patent name is " CRISPR-Cas9 specific knockdown people PD1
The Chinese patent application of the method for gene and the sgRNA " for selectively targeted PD1 gene；
1.1 take 1 × 10 respectively6To 5 × 107A cell, 4200 revs/min of centrifuge cell suspension 2min collect cell,
Fall supernatant liquid.
1.2 are added 1 × PBS 500 μ l, the 4200 revs/min of centrifuge cell suspension 2min of 4 DEG C of pre-coolings, collect cell,
Fall supernatant liquid, 200 μ l PBS are added and dissolve cell；
1.3 are added 25 μ l OB protease lysates, mix；
1.4 are added 220 μ l BL Buffer；
1.5 70 DEG C of water-bath 10min；
1.6, which are added 220 μ l dehydrated alcohols, mixes；
1.7 are put into column is collected in 2ml collecting pipe, and the liquid in step 1.6 is added and is collected in column；
1.8 12000 revs/min, it is centrifuged 1min, outwells waste liquid in collecting pipe；
500 μ l HBC Buffer are added in 1.9 collection columns, 12000 revs/min, is centrifuged 1min, outwells in collecting pipe and give up
Liquid；
700 μ l DNA Wash Buffer are added in 1.10 collection columns, 12000 revs/min, is centrifuged 1min, outwells collection
Waste liquid in pipe repeats this step operation 2 times；
Pipe is collected by centrifugation in 1.11 12000 revs/min of skies, is centrifuged 2min；
1.12 are added 100 μ l ddH in centrifugal column2O (70 DEG C of preheatings)；
1.13 stand 2min, 12000 revs/min, are centrifuged 2min, collect DNA, measure concentration, -20 DEG C save backup；
Step 3: the PCR amplification in site of missing the target:
PCR reaction system (25 μ l): 0.5 2.5 2 μ l of μ l, dNTP of μ l, 10 × Buffer of Transtar taq, forward direction are drawn
1 μ l of object (10 μM), 1 μ l of reverse primer (10 μM), 1 μ l of DNA, nuclease-free water add to 25 μ l.Amplification condition: 95 DEG C of 5min, 95
DEG C 30s, 68 DEG C of 30s (- 1 DEG C/circulation), 72 DEG C of 30s, 10 circulations, 95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 25 circulations, 72
℃5min；
Step 4: high-flux sequence: PCR product is constructed using VAHTS Universal DNA Library Prep Kit
Then library carries out high-flux sequence using Illumina 150PE.High-flux sequence strategy includes: purified genomic dna, is beaten
Disconnected genomic DNA, end are repaired, 3 ' ends plus A tail, two end adapter of join index, validate library and
Cluster, sequencing；
Step 5: data analysis: pcr amplification product sequence is obtained by the online primer blast of NCBI, and as customized
With reference to genome；Image base identification (Base is carried out using software CASAVA (v1.8.2) to sequencing result raw image data
Calling), preliminary quality is analyzed, and obtains the initial data (Pass Filter Data, PF) of sequencing sample；It uses
Cutadapt (1.9.1 version) optimizes initial data；The data after optimization are merged using Pandaseq (2.7 editions)；It will
Sequence after merging is compared with the customized reference genome using BWA software.Then detected using samtools
SNP/InDel；It is more believable in order to obtain that as a result, having carried out preliminary screening to result, filter condition is substrate quality value > 20,
Overburden depth > 4 map mass value > 40；
The result shows that all 6 potential sites of missing the target do not find insertion, missing and point mutation.
Embodiment 2:
People's PD1 gene C RISPR/Cas9 gene editing sgRNA2 misses the target site primer:
In addition to table 1 is changed to table 3 in comparative example 1, table 2 is changed to other than table 4, remaining step is all the same.
3 people's PD1 gene C RISPR/Cas9 gene editing sgRNA2 of table miss the target site screening
3 underscore base of table indicates mispairing
Table 4 misses the target site PCR primer information
The result shows that remaining potential site of missing the target is without insertion, missing other than OT7 and OT13 discovery single base mutation
And point mutation.There are identical point mutation for sample standard deviation after sequence alignment finds non-editing sample and editor, and mutational site does not exist
It is potential to miss the target in region, as illustrated in fig. 1 and 2, therefore show that undershooting-effect is not present in the sgRNA.
Embodiment 3:
People's PD1 gene C RISPR/Cas9 gene editing sgRNA3 misses the target site primer:
In addition in comparative example 1 step 1 using seqmap prediction miss the target site be changed to using CRISPOR prediction miss the target
Site, table 1 are changed to table 5, and table 2 is changed to other than table 6, remaining step is all the same.
5 people's PD1 gene C RISPR/Cas9 gene editing sgRNA3 of table miss the target site screening
5 underscore base of table indicates mispairing
Table 6 misses the target site PCR primer information
The results show that finding that three editing samples have the missing (as shown in Figure 3) of 3 bases in OT16.
Embodiment 4:
People's PD1 gene C RISPR/Cas9 gene editing sgRNA4 and sgRNA5 miss the target site primer:
The present embodiment using seqmap prediction site of missing the target is changed to and is missed the target site using CRISPOR prediction in addition to step 1,
Table 1 is changed to table 7, and table 2 is changed to other than table 8, remaining step is same as Example 1.
7 people's PD1 gene C RISPR/Cas9 gene editing sgRNA4 and 5 of table miss the target site screening
Table 8 misses the target site PCR primer information
It misses the target the result shows that hPD1-sg4 and hPD1-sg5 is not present in the site of missing the target of detection.
Comparative example:
The present embodiment is using 5 sites of missing the target described in T7EN1 enzyme cutting method detection embodiment 3:
Site information miss the target with table 5, PCR amplification primer is shown in Table 9.
9 people's PD1 gene sgRNA3 T7EN1 digestion PCR amplification primer of table
Step 1: miss the target site estimation and digestion PCR amplification design of primers and synthesis.PD1 used is predicted using CRISPOR
The potential site of missing the target of gene sgRNA3.5 potential sites of missing the target of screening scoring highest (possibility of missing the target highest), position of missing the target
Point information is as shown in table 5.The T7EN1 digestion pcr amplification product of product length about 740bp is devised for these sites of missing the target
(table 9) delivers company's synthesis；
Step 2: the selection and extraction of genome sample.Test procedure is the same as step 2 in embodiment 1；
Step 3: the PCR amplification in site of missing the target:
PCR reaction system (25 μ l): 12.5 μ l of Premix Taq (TaKaRa), 5 Enhancer μ l, 1 μ l of forward primer
(10 μM), 1 μ l of reverse primer (10 μM), 1.5 μ l of DNA, nuclease-free water add to 25 μ l.Amplification condition: 94 DEG C of 5min, 94 DEG C
30s, 60 DEG C of 30s, 72 DEG C of 30s, 34 circulations, 72 DEG C of 10min, 16 DEG C of preservations.
Step 4: the recycling of PD-1 gene PCR product carries out Cleanup (PCR Cleanup Kit)
After the mixing of EP Guan Zhongyu PCR product is added in the 4.1 CP Buffer for drawing 4~6 times of PCR product volumes, it is transferred to suction
In attached column (covering in 2ml collecting pipe), room temperature, 12000 revs/min of centrifugation 1min abandon filtrate；
4.2 700 μ l DNA Washing Buffer are added into adsorption column, and room temperature, is centrifuged 1min by 12000 revs/min,
Discard filtrate；
4.3 700 μ l DNA Washing Buffer are added into adsorption column, and room temperature, is centrifuged 1min by 12000 revs/min,
Discard filtrate；
4.4 by 12000 revs/min of adsorption column, sky centrifugation 2min, to exclude residual liquid.
4.5 will adsorb column sleeve in another new collecting pipe, with 70 DEG C of 40 preheated μ l ddH2O dissolution, room temperature are quiet
2min is set, 12000 revs/min, is centrifuged 1min；
4.6 collect obtained filtrate, with micro UV spectrophotometer measuring PCR product concentration and are marked, 2-8 DEG C
It saves backup, the normal phase, which saves, need to be put in -20 DEG C；
Step 5: PCR product T7EN1 digestion
5.1 digestion system
Positive control: 1.1 4.4 μ l of μ l, ddH2O of positive 5 μ l, 10 × T7EN1 Buffer of target PCR product, total volume 10.5
μl；
Sample to be tested: 5 μ l, 10 × T7EN1 Buffer of sample PCR product, 1.1 4.4 μ l of μ l, ddH2O, total volume 10.5
μl；
5.2 annealing
500ml water is added in the beaker of 1L the EP pipe in step 5.1 is placed in kickboard and is put after heating makes it boil
Enter in water, stops heating, room temperature is placed the beaker, to which its cooled to room temperature can (it is small that this process takes around 1 to 1.5
When)；
5.3 digestion
Above-mentioned reaction system is separately added into 0.5 μ l T7EN1 enzyme, after 37 DEG C of reaction 30min, at once plus 2 μ l DNA 6 ×
Loading Buffer boils 10min for 65 DEG C after mixing；
5.4 electrophoresis
The agarose gel electrophoresis for running 2% tests and analyzes digestion result；
As a result, site does not occur digestion band as shown in figure 4,5 predictions are missed the target, do not detect to miss the target.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
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<400> 8
gcctagtact tgcatccaga agg 23
<210> 9
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ggaggccagg taggtacatg gaa 23
<210> 10
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
cggcatgact ggtgacatat gga 23
<210> 11
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
gcgaggtgga gagagcctca 20
<210> 12
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cctttaagtt ccctggtcgt gtc 23
<210> 13
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cgtttcttcc ttgtcgctct tgg 23
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gtgctaagcc cgtcattgcc 20
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
cacttgagga gcccttcagc 20
<210> 16
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
caccaattga tccatgatcg gct 23
<210> 17
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ggagtggatg agcctctcca tga 23
<210> 18
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
gctcaacatg acactgcagg ga 22
<210> 19
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gtgagctgtg atcaccccac t 21
<210> 20
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ggcagttgtg tgacacggaa g 21
<210> 21
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
cctgttgtgt gacacagaag 20
<210> 22
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
gtggttgtgt gacacggaat 20
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
gcagttgtgt ggaatggaag 20
<210> 24
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
gcagttgtgt gacagcaaag 20
<210> 25
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
gcacttgtga gacactgaag 20
<210> 26
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
gaagttgtgt gacatggcag 20
<210> 27
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ggacttgggg gacacggaag 20
<210> 28
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
acacatgtgt gacacggaag 20
<210> 29
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
gcagtgttgg gacacggaag 20
<210> 30
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
gcaactaggt agatctggcc ttt 23
<210> 31
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gaataaggct gccaggtgct 20
<210> 32
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
gctaaggatc cagccagaga tca 23
<210> 33
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
gcagttggta tgtgaattcc att 23
<210> 34
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
ggtaatgggt taaaggcatg aga 23
<210> 35
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
gcaagacatc atgaaacact caa 23
<210> 36
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ggcttggcca cattcttatt gtt 23
<210> 37
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
tatcagcagt gtcaccctca tgt 23
<210> 38
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
gaaatttgtt caaagggtcg cct 23
<210> 39
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
gaaacctgac ccttgttagg cag 23
<210> 40
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
gcgtatcagg tcatgtcatg ttc 23
<210> 41
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
cagatgggca aaggtaaaac cat 23
<210> 42
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
ctgttcctct gctcaaaacc ctc 23
<210> 43
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
gcatgtgaag tgcctagaat gct 23
<210> 44
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
gactgtcagt gcaaggagag gaa 23
<210> 45
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
ctcttgctct aggcatcact gca 23
<210> 46
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
ggtattggaa actggggagg g 21
<210> 47
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
ggacgtgctg aatagttcac agt 23
<210> 48
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
aggtgccgct gtcattgcgc 20
<210> 49
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
aggtgctgct gtcattgctc 20
<210> 50
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
aggtgctgct accattgtgc 20
<210> 51
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
aggtgacgct attattgctc 20
<210> 52
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
aggtgatgat gtcattgagc 20
<210> 53
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
aggtggccct gacattgccc 20
<210> 54
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
caggaggagc tgacactgtc 20
<210> 55
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
aaagccacac tcgggaacaa 20
<210> 56
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
caacctgttt gcctggacac 20
<210> 57
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
taggatgggc acagactgga 20
<210> 58
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
ttttcgggat gacgtggcag 20
<210> 59
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
tgggaatgat gaacgttggg t 21
<210> 60
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
taatgggaag gtgggagcag 20
<210> 61
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
accttaattg ctgttttgga gaca 24
<210> 62
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
tccagggagt gacatcaggg 20
<210> 63
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
gtgtaggtgg ccctgacatc 20
<210> 64
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
gtggtgaccg aaggggaca 19
<210> 65
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
ggatgacgtt acctcgtgcg 20
<210> 66
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
cagtgcctgg acattcacc 19
<210> 67
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
aaaacccagt cccatccc 18
<210> 68
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
ggggaggtca caagccaca 19
<210> 69
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
ctctttcata aggaagggtc agg 23
<210> 70
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
gctgggatta caggcgtgag 20
<210> 71
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
gacggagtct tgctctgttg c 21
<210> 72
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
atctaatact ggcagaagca 20
<210> 73
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
tctccctgta gccgaggt 18
<210> 74
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
ctccgggtat cctgcaag 18
<210> 75
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
atccagggag tgacatcagg 20
<210> 76
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
gtctgggcgg tgctacaact 20
<210> 77
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
ttctggctag tgctacaact 20
<210> 78
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
gtctggctgg tactacaaca 20
<210> 79
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 79
ttctgggtgg agatacaact 20
<210> 80
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 80
atctgggctt tgatacaact 20
<210> 81
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 81
gactggccag ggcgcctgt 19
<210> 82
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 82
ggaatcgcct gggcgcctgt 20
<210> 83
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 83
agcctggcca aggcacctgt 20
<210> 84
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 84
ggactggcca aggagcctgt 20
<210> 85
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 85
tgactgacaa gggagcctgt 20
<210> 86
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 86
tgattggcca gtgtgcctgt 20
<210> 87
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 87
tctgaatctg aagaccagct gt 22
<210> 88
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 88
ctggccacat tctagacgat ga 22
<210> 89
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 89
tgccaatcat gggaaagtca g 21
<210> 90
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 90
ttgagtagcg ccaaaaggca 20
<210> 91
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 91
gtggtctgag cctcaaagca 20
<210> 92
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 92
ctgctcccgg tgctagaaaa 20
<210> 93
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 93
gtgactacca gacaaccagc t 21
<210> 94
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 94
tgctttctgc caaaagagat tca 23
<210> 95
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 95
ttccttgcac acgtcaggg 19
<210> 96
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 96
cgcctgctgt ccggtttaat 20
<210> 97
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 97
atcgagaatg agctgtgggc 20
<210> 98
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 98
tgcctaaaac ctgagcccag 20
<210> 99
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 99
agtggtggct cagcatcaaa 20
<210> 100
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 100
ccactgtcca tggcctcatt 20
<210> 101
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 101
cttgagtctg gctggaaggg 20
<210> 102
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 102
gctgctgctg actaaagctg 20
<210> 103
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 103
tccactagag gaacaggacg a 21
<210> 104
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 104
ccgtgttctc cttcctccac 20
Claims (10)
1. it is a kind of detect CRISPR-Cas9 undershooting-effect method, which is characterized in that itself the following steps are included:
A. it predicts the site of missing the target of sgRNA, and obtains PCR amplification primer；
B. cdna sample is chosen, the cdna sample includes gene editing sample and do not carry out gene editing sample；
C. PCR amplification is carried out to the site of missing the target that step (a) predicts；
D. high-flux sequence is carried out to the resulting PCR product of step (c)；
E. interpretation of result, sequence alignment.
2. the method for detection CRISPR-Cas9 undershooting-effect according to claim 1, it is characterised in that: in step (a),
The method in the site of missing the target of the prediction sgRNA is to be predicted by software or website.
3. it is according to claim 2 detection CRISPR-Cas9 undershooting-effect method, it is characterised in that: the software or
Person website is selected from seqmap, CRISPOR, CRISPR finder, CRISPR Design, sgRNAcas9, CRISPRdirect,
Any in COSMID, Off-Spotter, E-CRISP.
4. the method for detection CRISPR-Cas9 undershooting-effect according to claim 2, it is characterised in that: by described soft
Part or the method for website prediction are the highest at least 3 potential sites of missing the target of screening scoring.
5. the method for detection CRISPR-Cas9 undershooting-effect according to claim 1, it is characterised in that: in step (c),
PCR reaction system are as follows: Transtar taq 0.5 μ l, 10 × Buffer 2.5 μ l, dNTP 2 μ l, 1 μ l of forward primer (10 μM),
1 μ l of 1 μ l of reverse primer (10 μM), DNA, nuclease-free water add to 25 μ l；Amplification condition: 95 DEG C of 5min, 95 DEG C of 30s, 68 DEG C
30s (- 1 DEG C/circulation), 72 DEG C of 30s, 10 circulations, 95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 25 circulations, 72 DEG C of 5min.
6. the method for detection CRISPR-Cas9 undershooting-effect according to claim 1, it is characterised in that: in step (d),
High-flux sequence strategy includes: purified genomic dna, interrupts genomic DNA, and end is repaired, 3 ' ends plus A tail, join index
Two end adapters, validate library and cluster, sequencing.
7. the method for detection CRISPR-Cas9 undershooting-effect according to claim 1, it is characterised in that: in step (e),
The method of interpretation of result are as follows: pcr amplification product sequence is obtained by the online primer blast of NCBI, and refers to base as customized
Because of group；The identification of image base is carried out using software CASAVA to sequencing result raw image data, preliminary quality analysis is surveyed
The initial data of sequence sample；Optimize initial data using Cutadapt；The data after optimization are merged using Pandaseq；
Sequence after merging is compared with the customized reference genome using BWA software；Reuse samtools detection
SNP/InDel。
8. the method for detection CRISPR-Cas9 undershooting-effect according to claim 7, it is characterised in that: result carries out just
Step screening, filter condition are substrate quality value > 20, and overburden depth > 4 map mass value > 40.
9. the method for detection CRISPR-Cas9 undershooting-effect according to claim 1, it is characterised in that: detection people PD1 base
Because CRISPR/Cas9 gene editing sgRNA1 miss the target site when, the resulting PCR amplification primer of step (a) have table 2 shown in sequence
Column；Detection people PD1 gene C RISPR/Cas9 gene editing sgRNA2 miss the target site when, the resulting PCR amplification primer of step (a)
With sequence shown in table 4；Detection people PD1 gene C RISPR/Cas9 gene editing sgRNA3 miss the target site when, step (a) institute
The PCR amplification primer obtained has sequence shown in table 6.
10. the method for detection CRISPR-Cas9 undershooting-effect according to claim 1, it is characterised in that: detection people PD1
Gene C RISPR/Cas9 gene editing sgRNA4 and sgRNA5 miss the target site when, the resulting PCR amplification primer of step (a) has
Sequence shown in table 8.
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| CN113005185A (en) * | 2019-12-20 | 2021-06-22 | 青岛华大基因研究院 | Method for detecting gene editing efficiency and off-target in cell |
| CN111893170B (en) * | 2020-08-07 | 2022-08-19 | 珠海舒桐医疗科技有限公司 | Method for detecting CRISPR-Cas off-target effect in whole genome range in vitro |
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| CN114807333A (en) * | 2022-05-31 | 2022-07-29 | 华南农业大学 | Method for identifying whole genome variation of gene editing animal |
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| CN112750499A (en) * | 2021-01-19 | 2021-05-04 | 无锡市第五人民医院 | Method and system for improving safety of gene editing technology |
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