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Definitions

• This disclosure relates to methods, compositions, and kits and systems that can be used in DNA modification, including DNA sequence knock-in or knock-out, DNA mutation, DNA epigenetic modification, chromatin modification in a DNA sequence-specific manner, and other types of genome editing. More specifically, this disclosure relates to methods that can deliver the system of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components, mutations, fusions, and variations thereof, without the use of any carrier vector.
• CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
• This invention specifically teaches a process of editing a genome with specificity and precision that permits substituting a single nucleotide, including host cells as challenging as a pluripotent stem cell.
• a novel method for highly efficient DNA sequence alterations.
• the method can be used to edit chromosomes, to engineer cellular markers through insertion of genes, or to create epigenetic changes by using cas ⁇ -enzyme fusions where the enzymes can be DNA epigenetic modifying enzymes or chromatin modifying enzymes, etc.
• the novel technology also differs from all previously known technologies in that the
• CRISPR/CAS system can function in ways that are“clean”, i.e. they have not been in contact with any virus, or are carried DNA molecules that can insert into the chromosome in unintended locations. It is also noted that the disclosed system can generate previously unattainable efficiency while keeping off-target changes to the minimum. Utility of the invention can be found in virtually all areas that involve DNA editing or epigenetic modification. In contrast, the 8,697,359 patent does not teach how to provide a system where CRISPR/Cas can be efficiently attained in eukaryotic cells while minimizing the potential problem of unintended genome changes.
• the current disclosure provides an RNA-based system that provides both the Cas enzymes and guide RNAs, and in cases involving DNA break repair, a“patch” template RNA or DNA, all without the need of any exogenous DNA molecules (except when a DNA template is a preferred template for DNA break repair).
• the all-RNA CRISPR/Cas (as used herein, the term“all-RNA” primarily refers to the delivery of the components of a
• CRISPR/Cas machinery and does not exclude DNA as template) system disclosed herein does not require any viral elements that may create problems for human clinical use of the process or resulting cells.
• This system can be provided as methods, processes, or reagent kits to achieve gene disruption through CRISPR/Cas-promoted indels, genome sequence editing to the precision of a single base, or gene replacement through break repair and replace after CRISPR/Cas treatment in cultured cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), at enhanced efficiency and specificity compared to what has been shown in the field.
• ESCs embryonic stem cells
• iPSCs induced pluripotent stem cells
• An important aspect of the current disclosure is the use of an all-RNA delivery method to enable a polynucleotide-guided genomic cutting system in eukaryotic cells, with designs particularly useful in mammalian cells, and a process empirically developed for difficult-to-maintain cells such as pluripotent stem cells, which easily escape the pluripotency state if perturbed.
• the disclosed method will also work in tissue stem cells such as, without limitation, neural progenitor cells, oligodendrocyte progenitor cells, mesenchymal stem cells, hematopoietic stem cells, etc.
• RNA as in vitro transcribed (IVT) RNA and the Cas enzyme as mRNA using common nucleoside triphosphates (NTP) or NTP with chemical modifications.
• IVT in vitro transcribed
• NTP common nucleoside triphosphates
• the high level of enzyme activity can be concentrated within a short-time window in a highly controllable fashion.
• the transient nature of RNA-mediated high enzyme expression level provides an ideal composition for the purpose of chromosomal modification.
• the short-burst enzyme expression provides additional benefits in reducing off-target effects because long existence of the enzyme, such as that from plasmid DNA vectors or integrated viral vectors can result in continued off-target effects.
• the gRNA is delivered at various ratios to Cas mRNA, sometimes involving multiple delivery via transfection. Because once mRNA of cas is translated into Cas protein, the protein is likely to have a longer half-life than mRNAs and gRNA.
• the disclosure herein demonstrates that, by adjusting the gRNA amount, which can also be referred to as gRNA/c v mRNA ratio, the process can result in precise, single base editing, in addition to more commonly seen longer inserts or deletions or rearrangements of the chromosome.
• Example 4 of the current disclosure demonstrates the increased precision of the disclosed methods by showing a successful example of how a single base on the chromosome can be changed using the all-RNA methodology in a human iPSC clone.
• RNA itself can be highly immunogenic (Kawai and Akira, 2007;
• Mammalian cells are equipped with a battery of sensors that can detect exogenous RNA and activate antiviral defense pathways which prime cytostatic and apoptotic pathways and alert neighboring cells to the very same stimuli via excreted signals such as interferon alpha and beta.
• the more broadly-expressed sensors such as TLR3 and RIG-I primarily detect double-stranded RNA (the production of dsRNA being a distinctive feature in many viral life cycles) but can also be activated by synthetic mRNA (Kormann et ah, 2011).
• Technical means were found to minimize immunogenic responses to synthetic mRNA during the course of iPSC generation with mRNA (Warren et ah, 2010).
• the delivery of the all-RNA CRISPR/Cas system into human cells was accompanied with the addition of B18R.
• the RNA molecules can be delivered into human or non-human cells when the RNA molecules are sufficiently purified to remove aberrant transcripts during in vitro transcription.
• the delivered RNA molecules are modified to evade cellular immune detection.
• the novel CRISPR/Cas system provides technical enablement for genome engineering in these aspects: polynucleotide-guided, without the requirement of protein engineering for each target site; fully controlled process through RNA delivery that does not leave a genomic footprint; easy to achieve desired modification efficiency in different cell types by varying treatment time; higher success rate and lower off-target effects than ZFN or TALEN or previously reported CRISPR/CAS methodologies because of the designed higher enzyme activity in a shorter time window; precise genome modification in a highly efficient process that can be performed in pluripotent stem cells without perturbing the stem cell state, enabled at least in part by a previously unknown and nearly uncontrollable factor— the gRN A/c v-m RNA ratio, which is not optimal if plasmids, viral vectors, and
• ribonucleoprotiens are used. Compared to recently published CRISPR/CAS systems, the disclosed all-RNA format uniquely enables minimization of unwanted chromosomal changes.
• the method disclosed herein is based on the unexpected benefits of adjusting doses of gRNA and CAS enzyme via cas mRNA.
• the mRNA that encodes Cas9 and sgRNA contains a 5’diguanosine cap and poly(A) tail, and modified nucleotides that make the mRNA less toxic to a cell.
• the modified nucleotides comprise 5-methyl-Cytosine, 2-Thio-Uracil, or pseudouracil.
• the mRNA encoding Cas9 is given together with B18R.
• mutated forms of Cas9 protein that contain mutations to one or both of their endonuclease genes.
• Applicant have produced three non-naturally occurring mutant Cas9 proteins with mutations to their endonuclease active site. These mutant Cas9 proteins are encoded by SEQ ID NOS:
• a non-naturally occurring CRISPER-Cas system comprising an mRNA that encodes for a mutated Cas9 protein that has a mutation in its endonuclease active site and at least one mRNA that encodes for a guide RNA that upon entry into a cell produces the mutated Cas9 protein and guide RNA.
• the Cas9 protein and guide RNA targets and hybridizes to a target sequence of a DNA with a single point mutation that upon action of the mutant Cas9 protein and guide RNA corrects the mutation in the target sequence.
• RNA that encodes Cas9 and sgRNA contains a 5’diguanosine cap and poly(A) tail.
• a template to facilitate DNA break is also provided.
• the template can be a double-stranded DNA molecule or single-stranded DNA molecule.
• the template is an RNA molecule.
• the Cas9 bears a mutation that disrupts one of the two endonuclease active sites.
• the Cas9 protein mutants are encoded by SEQ ID NO: 2, or SEQ ID NO: 3.
• Cas9 protein mutant has mutations in both endonuclease active sites and is encoded by SEQ ID NO: 4.
• Cas9 is fused to another enzyme that can alter epigenetic markers on either the DNA or chromatin proteins.
• the molar ratio between Cas9 mRNA:sgRNA is between 1 : 1,000 to 1,000: 1. In some embodiments of the method, the molar ratio between Cas9 mRNA:sgRNA is between 1 : 1,000 to 1,000: 1.
• the molar ratio of Case9mRNA: sgRNA is 1:1,000; 1:950; 1:900; 1:850; 1:800; 1:750; 1:700; 1:650; 1:600; 1:550; 1:500, 1:450; 1:400; 1:350; 1:300; 1:250; 1:200; 1:150; 1:100; 1:50; 1:40; 1:30; 1:25; 1:20; 1:15; 1:10; 1:9; 1:8; 1:7; 1:6; 1:5; 1:4.75; 1:4.5; 1:4.25; 1:4; 1:3.75; 1:3.5; 1.3.25; 1:3; 1:2.9; 1:2.8; 1:2.75; 1:2.7; 1: 2.6; 1:2.5; 1:2.4; 1:2.3; 1:2.25; 1:2.2; 1:2.1; 1:2; 1:1.9; 1:1.8; 1:1.7; 1:1.6; 1:1.5; 1:1.4; 1:1.3; 1:1.2; 1:
• the repair template is localized to the DNA break site through fusion to the sgRNA as on one molecule. In some embodiments, the repair template is localized to the DNA break site through fusion to an aptamer that binds Cas9.
• the precision-enabling nature of the disclosed methods makes the disclosed technology most suitable for creating cells for treating human diseases, such as without limitation, Methylmalonyl-CoA mutase deficiency, 3-Methylcrotonyl-CoA carboxylase deficiency, Gaucher’s disease, Ogden syndrome, Lesch-Nyhan syndrome, Leigh disease, pyruvate dehydrogenase deficiency, 3 -hydroxy-3 -methyl glutaryl-CoA lyase deficiency, carboxylase deficiency, multiple, late-onset, fumarase deficiency, fibrodysplasia ossificans progressive, n-glycanse 1 deficiency, siderius type X-linked mental retardation,
• human diseases such as without limitation, Methylmalonyl-CoA mutase deficiency, 3-Methylcrotonyl-CoA carboxylase deficiency, Gaucher’s disease, Ogden
• phenylketonuria tay-sachs disease
• alpha-galactosidase A deficiency sickle cell anemia
• maple syrup urine disease alpha-galactosidase A deficiency
• FIG. 1 Creation of IVT templates for generating cas9 mRNA sgRNA 2% agarose gel shows bands of purified linearized DNA generated by cutting cas9 or sgRNA gene encoding plasmids with restriction enzymes.
• Figure 2 mRNA encoding the Cas9 enzyme and sgRNA against fluorescent protein mWasabi.
• 2% agarose gel shows band of the cas9 mRNA with poly(A) tails and the sgRNA against mWasabi.
• Figure 3 Effects of disrupting the expression of mWasabi gene integrated in to the chromosomes of human 293 cells. Constant amount of cas9 mRNA and increasing amount of sgRNA was delivered into 293-mWasabi cells in a single transfection. The control well did not receive any RNA but was treated with the same transfection reagents.
• Figure 4 Examples of using all-RNA CRISPR/CAS system in creating a mutation in human gene.
• Each dsDNA break point can be directed by a pair of sgRNAs.
• a sequence replacement can be made with either one or two break points as shown in the figure. When 4 sgRNAs are relied upon to direct the replacement, the specificity is maximized.
• Figure 5 Examples of using all-RNA CRISPR/CAS system in creating a mutation in human gene with a dimerized Cas9 enzyme encoded by modified mRNA.
• the CRISPR/CAS mediated genome editing specificity can be further enhanced with dimerizing Cas9, particularly when delivered through encoding mRNA.
• Other domains can be fused to Cas9 in a similar fashion for epigenetic modifications.
• Figure 6 Primer design for qPCR. This design enabled detection of a single base change in iPSCs by real time PCR
• Figure 7 Example of Amplification Ct curves. This curve shows how mutation rate at a given location on the chromosome was detected by well-designed qPCR.
• FIG. 8 Sample amplification plot for clonal amplicon library screening. qPCR screening of clonal amplicon libraries commonly result in high variation, however given a bulk population that has an HDR efficiency of ⁇ l%, there will be a small number of low Ct outlier wells. Once left shifted Ct outliers were identified, and the corresponding wells were expanded in duplicate plate.
• Figure 9 Sample chromatograms for clonal amplicon library screening. A single base switch from T to G was achieved in a single iPSC clone which is heterozygous for the intended MEF2C locus.
• RNA molecules for CRISPR/CAS were used, but only in fertilized animal eggs or embryos through microinjection, to various results (Wu et al. Cell Stem cell , Volume 13, Issue 6, 5 December 2013, 659-662; Liang et al. Protein & Cell , May 2015, Volume 6, Issue 5, pp 363-372;
• mRNA-based encoding wild-type cash from different bacteria species e.g. Streptococcus pyogenes , Streptococcus mutans .
• Campylobacter jejuni N meningitidis , Escherichia coli , Francisella novicida , and other species known to contain type II CRIPSR system (Fonfara et al., 2013).
• the genes of such Cas9 enzyme, or other Cas enzymes can be cloned from either the bacterial genomic DNA or cDNA using cloning techniques known in the art.
• a cas9 gene is cloned behind a promoter, such as that of bacterial phage T7 RNA polymerase, T3 RNA polymerase, or Sp6 RNA polymerase, or other RNA polymerases.
• the cassette that encompass the promoter, the cas9 coding DNA, a fragment that encodes a poly(A) tail to mRNA suitable for the stability and expression in eukaryotic cells can be used for in vitro translation (IVT) as a linear template or cloned into a vector such as a plasmid, a phagemid, or other carriers of DNA sequences (for example Figure 1).
• IVTT in vitro translation
• mRNA is produced by in vitro transcription under optimized conditions as described herein.
• An embodiment of the disclosure are synthetic mRNA transcripts that serve as efficient templates for translation in living cells by incorporating a 5’ diguanosine cap and a poly(A) tail.
• the cap and tail can be incorporated into IVT transcripts enzymatically or co-transcriptionally.
• Benefits of enzymatic capping include high RNA yields, low costs, and a potential for producing almost pure capped RNA.
• a synthetic cap analog is included at high concentration in the IVT reaction buffer, the cap being
• RNA polymerase a polyadenylation polymerase
• in vitro transcription is preferably carried out with modified nucleotide triphosphates (NTPs), such as 5-methyl-Cytosine, 2-Thio-Uracil, or pseudouracil, or other modified nucleotides able to substitute unmodified nucleotide in RNA molecules that do not significantly alter the RNA’s functions.
• NTPs modified nucleotide triphosphates
• Using modified nucleotides help reduce cellular immune response, which is particularly important when repeated deliveries of mRNA into the host cells are required to achieve desired level of genome modification among host cells, or the host cells are hypersensitive to exogenous RNA molecules.
• the current disclosure further relates to generation of sgRNAs.
• sgRNAs as guide for CRISPR/CAS are introduced via a DNA vector or viral vector, whereby sgRNA-encoding DNA is placed behind a promoter that can drive transcription of short RNAs, e.g. U6 or Hl promoters.
• sgRNA encoding DNA is placed behind a promoter that is suitable for in vitro transcription, e.g. a T7, T3, or Sp6 promoter ( Figure 1).
• the cassette that encompasses the promoter and the sgRNA coding DNA can be used as a linear template or cloned into a vector such as a plasmid, a phagemid, or other carriers of DNA sequences.
• a transcription termination can also be achieved by having a transcription terminator sequence.
• a vector is the pIVT plasmid described previously (Warren et al., 2012).
• sgRNAs are created by IVT using modified or unmodified NTP ( Figure 2).
• the wildtype Cas9 enzyme naturally has two endonuclease functional domains SEQ ID NO: 1.
• the Cas9 enzyme can be converted from a dsDNA cutting enzyme into a single-strand DNA (ssDNA) nicking enzyme, e.g. SEQ ID NO: 2, SEQ ID NO: 3.
• a double-stranded break can still be created, but as opposed to a double- stranded break created by a wildtype Cas9, two sgRNAs are needed, thereby providing added sequence-specificity to the process ( Figure 4).
• mRNA is created to express such mutants of Cas9 that nicks one strand when guided by one sgRNA.
• the cas9 mRNA encodes a version of Cas9 further mutated to remove both of its endonuclease domains (SEQ ID NO: 4) and fused to an artificial nuclease domain such that of restriction enzyme Fokl or other such restriction enzymes (Figure 5).
• the resulting mutant form of Cas9 needs to form a dimer to function as an endonuclease, requiring the target sites defined by the pair of sgRNA sequences to be close together, preferably with a distance between about 5-30 or about 10-20 nucleotides (nts), or about between 12-18 nts, providing further specificity.
• Another aspect of the current invention relates to the selection of
• CRISPR/CAS target sites The design of a preferred sgRNA matching site on a eukaryotic genome is well established.
• a chromosomal knock-in process in order to maximize target specificity during a chromosomal knock-in process (replacing a segment of sequence, which can be as short as a single nt, of the chromosome with another by providing a DNA template), it is hereby disclosed that two double-stranded cuts are made by using either nicking Cas9 mutants or a Cas9-Fokl fusion when choosing the target sites.
• An example is illustrated in Figure 4.
• the Cas9 or its nicking or blunt mutants is in- frame fused to epigenetically modifying enzymes, such as protein arginine methyltransferases PRMT1 and PRMT4 (CARM1), DNA methyltransferases, histone methyltransferases, histone acyltransferases etc.
• epigenetically modifying enzymes such as protein arginine methyltransferases PRMT1 and PRMT4 (CARM1), DNA methyltransferases, histone methyltransferases, histone acyltransferases etc.
• RNA for providing sgRNA
• the guide RNA, structure RNAs as in a typical sgRNAs, and if necessary, a linker RNA can be further fused to a patch template RNA for local repair after cutting by Cas9 enzyme. It is known in the field that RNA can be used for homologous DNA break repair, which is incorporated herein by reference (Storici et al., 2007).
• a DNA or RNA aptamer that specifically binds to Cas9 is linked to a sequence replacement“patch” template in order to achieve gene knock-in or knock-out through the use of a template polynucleotide.
• the patch By physically being attached to the Cas9 enzyme, the patch can be delivered close to the site of CRISPR/CAS cutting.
• the patch template can be either DNA or RNA.
• RNA molecules do not need to translocate into the nucleus, thereby eliminating a bottleneck typically presented by nuclear entrance, as well as many layers of uncertainty in terms of molar ratio between DNA and mRNA.
• the Cas proteins can be highly expressed immediately after the encoding mRNA enters cytoplasm by a transfection or electroporation process.
• the RNA molecules naturally have a relatively short half-life, therefore making the control of off-target effects of the CRISPR/CAS system more manageable than using DNA vectors or viral vectors.
• a further embodiment of the current disclosure in relevance to dosing control relates to adjusting the ratio between gRNA Cas and mRNA. Because the dose of cas 9 mRNA can be essentially proportionally correlated to the level of Cas enzyme, the all-RNA CRISPR/Cas system disclosed hereby enables a direct matching between the two component of CRISPR/Cas, namely the Cas enzyme and the gRNA, in order to obtain the highest on- target and the lowest off-target DNA cutting.
• the DNA encoding Cas9 from bacterium Streptococcus pyogenes was codon maximized for optimal expression in mammalian, particularly human cells.
• the complete gene was assembled from 3 fragments generated through commercial gene synthesis service (Gene Oracle); mutations that disrupt DNA endonuclease domains were included during gene synthesis, resulting in different versions of cas9 as delineated in SEQ ID NOS: 1-4.
• Synthetic mRNA was generated in IVT reactions using a 4: 1 ratio of anti reverse cap analog (ARCA) to GTP to generate a high percentage of capped transcripts. Twenty percent substitution of 5m-CTP for CTP and 2-Thio-UTP for UTP in the nucleotide triphosphate (NTP) mix was employed to reduce the immunogenicity of the RNA products. ARCA and modified NTPs were purchased from Trilink Biotechnologies (San Diego). A 2.5x NTP mix was prepared (ARCA:ATP:GTP:C:5m-CTP:UTP:Pseudo-UTP at
• Each 20 pL IVT reaction comprised 8 pL NTP mix, 2 pL IOc T7 Buffer, 8 pL DNA template and 2 pL T7 enzyme (Promega). Reactions were incubated 4-6 hours at 37°C and then treated with 1 pL RNAse-free DNase for an additional 30 minutes at 37°C before being purified on a spin column, the RNA product being eluted in a volume of 80 pL.
• Synthetic sgRNA was generated in IVT reactions using a 4: 1 ratio of ARCA cap analog to GTP to generate a high percentage of capped transcripts. Twenty percent substitution of 5m-CTP for CTP and 2-Thio-UTP for EGTR in the nucleotide triphosphate (NTP) mix was employed to reduce the immunogenicity of the RNA products. Cap analog and modified NTPs were purchased from Trilink Biotechnologies. A 2.5x NTP mix was prepared (ARCA:ATP:GTP:C:5m-CTP:UTP:Pseudo-UTP at 15: 15:3.75:3:0.75:3:0.75 mM).
• Each 20 pL IVT reaction comprised 8 pL NTP mix, 2 pL IOc T7 Buffer, 8 pL DNA template and 2 pL T7 enzyme (Promega). Reactions were incubated 4-6 hours at 37°C and then treated with 1 pL RNAse-free DNase for a further 30 minutes at 37°C before being purified on a spin column, the RNA product being eluted in a volume of 80 pL. 3 pL Antarctic Phosphatase (New England Biolabs) was added for 10 min to remove immunogenic 5’ triphosphate moieties from uncapped transcripts and 10 pL of reaction buffer. Phosphatase reactions were incubated for 30 minutes at 37°C and the IVT products were repurified if necessary ( Figure 2).
• a complete all-RNA CRISPR/CAS system was created to disrupt a fluorescent protein (FP) mWasabi (Allele Biotech) permanently expressed in mammalian cell NIH-3T3.
• NIH3T3 -mWasabi cells were grown at 15% confluency in serum-free medium, cas9 mRNA and sgRNA were co transfected into the cells; after 2 hrs serum-containing medium was added.
• FP fluorescent protein
• mWasabi Allele Biotech
• top panels show where the cells are (phase contrast); bottom panels show the cells that are still fluorescent (green fluorescence channel).
• the three arrows in the right-bottom panel point to the cells that lost the green fluorescence in the well that received the higher dose of sgRNA together with cas9 mRNA. No cells in the 0 or 0.2 ng sgRNA wells lost the green fluorescence.
• EXAMPLE 6- Method embodiments for generating a single base pair mutation via an mRNA-based CRISPR/Cas9 system.
• a 300bp sequence surrounding the intended mutation site is run through the web-based sgRNA design tool. (“MIT Crispr Design Tool” MIT). 2) Guide RNA selection is determined by 2 parameters: a) proximity to intended mutation, and b) potential off target score. 3) A minimum of two sgRNA sites are selected. (Optimal parameters would be a PAM site within 5bp of intended mutation and a sgRNA score of >70.)
• oligonucleotide donor (ssODN) repair template oligonucleotide donor (ssODN) repair template
• the reverse Primer (mutation specific) can have the intended mutation at the 5’ leading end.
• IVTT in vitro transcription
• IVT template production of sgRNA 1) design and synthesize a forward primer with the following 3 elements: a) a T7 promoter, b) the protospacer element sequence (step A.I.2), and c) a crRNA specific sequence.
• a universal reverse primer (sgRNA_Rev) is used to complete the primer pair. 2), using these primers and the pT7sgRNA plasmid as a template, a PCR reaction is performed to create the IVT template ( ⁇ 13 lbp). Dpnl digesting the reaction sample and perform a PCR cleanup, so it can be suitable for the in vitro transcription reaction.
• IVT reaction to produce CRISPR elements 1) using the templates created via PCR, perform an IVT reaction to transcribe the sgRNA and Cas9wt mRNA. 2) Purify and QC transcripts via gel imaging and Bioanalyzer (Agilent).
• Plating of exemplary target cells 1) Cells are cultured in E8 media supplemented with ROCK Inhibitor (Y27632) during passages. 2) The day before transfection, cells are passaged into a 6-well plate at a density of 2.5xl0 5 cells/well.
• I. Exemplary method embodiments for lysis of treated cells and amplification of gDNA for screening 1) Performing lysis of leftover cells from D.II.5 experimental and negative control wells. Resuspending cells in Allele’s Mouse Tail lysis buffer (Allele Biotech, San Diego) and run samples using lysis program in thermocycler. The resulting lysate is amplified ( ⁇ 26 cycles) using primers designed in A.III.3 using Herculase II fusion DNA polymerase (Agilent Technologies). Performing PCR cleanup on the PCR product.
• the resulting experimental and negative control amplicon libraries is designated as the experimental and negative control“Bulk populations.” 2) ETsing the Nanodrop to quantify the PCR product, performing a dilution to standardize all amplicons to 60fg/pl.
• Performing lysis protocol (same as E.1.1) on source plate. Performing a test PCR on 3 wells with 2 lysate volumes to identify optimal lysate template volume. 2) Performing plate PCR of cell lysate from source plate. Once PCR is complete, run the PCR products from the plate on a large format agarose gel to confirm amplification and provide record for any variation in amplification yield. 3) LTsing the SurfaceBind Purification Plate (Allele Biotech), purifying PCR product according to protocol. Elute in 30m1 Elution Buffer. 4) Performing a 1 : 1000 dilution with the purified PCR product into molecular grade water. Lise 2ml collection plate to maintain plate format.
• the amplicon library is now at a suitable concentration for screening.
• 5) Performing SYBR green based Standard Quantification qPCR screening on amplicon libraries from the four 96-well plates.
• the most left-shifted qPCR Ct curves represent wells having the highest probability of containing a mutant cell population (i.e. with the intended HDR event).
• Sanger sequencing analysis on the original purified amplicon library stock corresponding with all outlier wells.
• sequencing results indicate a mixed population
• a second round of single-cell cloning is performed (repeat steps starting from E.III). After cells reach confluency in the l2-well plate (-10 5 cells), expand and freeze the confirmed outliers, and proceed to a second round of single-cell cloning. Recommended: lyse, amplify and sequence any remaining cells to test whether the mutation is preserved after passaging.
• b) When sequencing results indicate a pure population (i.e. the ratio of chromatogram peaks corresponding to WT and Mutant are 1 : 1 [indicating a heterozygous population]), confirm the cells are heterozygous by performing a second analytical round of single-cell cloning by analyzing 24 to 48 wells. Expanding cells to a 6-well plate format. Cryopreserve cells at a concentration of 10 6 cells/vial. Lyse, amplifying and sequencing a portion of the cells to test when mutation is preserved after passaging.
• df (Vfmal/Vinitial).
• Step 8.2.3 After performing the in vitro transcription reaction as completed in Step 8.2.3, add 2 m ⁇ of RQ1 RNase-free DNase to each reaction in order to remove the DNA template.
• Step 5 Incubate the mixture for at least 30 minutes at 37°C in the T100 Thermal Cycler. After the incubation period from Step 5.2.5 is complete, add 5 m ⁇ of 10X Antarctic phosphatase reaction buffer and 3 m ⁇ of Antarctic Phosphatase to each reaction.
• RNA bands with LED light Determine when a clear RNA band has appeared at the correct size position Cas9 size: ⁇ 4400nt; sgRNA size: ⁇ l50nt
• cell counts should be between 300,000 to 500,000 cells/ml. Carry out serial dilution to get a 2-3 cells/lOO pL concentration in Y27632 supplemented TeSRTM-E8TM.
• Corning® Matrigel® (0.5ml/well using 1 :80 dilution in DMEM).
• cryopreservation media Y27632 supplemented TeSRTM-E8TM, 10% HSA, and 10% DMSO
• Targeted loci are PCR amplified according to protocol outlined in VI.c. Template should be from lysate of non-transfected cells. In this case, forward and reverse primers should also have overlap regions with the pIVT vector for Gibson Assembly.
• pIVT vector must also be linearized via PCR by using pIVT-F and R pIVT-F: GCTGCCTTCTGCGGGGCTTGCCT (SEQ ID NO: 7)
• pIVT-R GGTGGCTCTTATATTTCTTCTTACTC (SEQ ID NO: 8)
• Gibson Assembly reaction is incubated for lhr at 50°C, then transformed into DH5a chemical competent cells.
• the resulting vector will be designated as the wild-type vector.
• -Forward primer should be around 200-300bp upstream of intended mutation.
• step VI.c With the amplicons produced in step VI.c, standardize the concentration down to 60fg/ul.
• step VIII.b.2. Assign amplicon standards in the orientation outlined in step VIII.b.2.
• A. In triplicate, assign the standardized amplicon libraries from bulk population into the plate. Make sure to include a negative control (ssODN only transfected cells).
• step VIII.b.4 Run software as outlined in step VIII.b.4. Before starting, make sure to assign CRISPR amplicon libraries and negative control from the experiment according to how they were allocated in the reaction plate in the“Plate Setup” window. Run program when everything is setup properly.
• amplicon libraries can be compared with each other (CRISPR treated cells and non transfected cells) and with the standards.
• ACt for CRISPR treated cells should be calculated by taking the negative control Ct value and subtracting the CRISPR treated cells Ct value.
• CRISPR treated cells should show a ACt comparable to the 1% standard. (Note: it has been observed that the ACt increases after one passage). 2. ) When the ACt for bulk population is ⁇ l%, proceed to single cell cloning procedure as outlined in step V
• step VIII.b.4 Run software as outlined in step VIII.b.4 with the“96_well_screen” template file (D: ⁇ Applied Biosystems ⁇ StepOne Software v2.3 ⁇ config ⁇ templates). (Make sure the“Run Setup” window and it’s parameters are identical with those in the bulk qPCR screen assay).
• SEQ ID NO: 1 cas9_wt

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