CRISPR-Cas9 HDR knock-in/precise editing
Materials Required
Principle
CRISPR-Cas9 HDR knock-in uses a guide RNA to direct Cas9 to a genomic target adjacent to a PAM, where Cas9 creates a double-strand break; if a donor DNA template with homology to the cut region is present, cellular HDR can copy the donor sequence into the genome, producing a precise substitution, tag, reporter, or insertion rather than an indel[1][2][3][4]. The readout is the fraction of alleles or cells carrying the intended donor-derived edit, measured by junction PCR, restriction-fragment analysis, Sanger sequencing, amplicon deep sequencing, flow cytometry for reporter knock-in, or clone genotyping; NHEJ indels and partial or non-HDR insertions are measured in parallel because they compete with or confound precise HDR outcomes[4][5][6][7].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use cultured mammalian cells, primary cells, embryos, or another validated sample system; Cas9 may be delivered as plasmid, mRNA, or preassembled Cas9 ribonucleoprotein, but RNP delivery has been used to obtain rapid editing and reduce prolonged nuclease exposure in human cells and primary immune cells[4][6][8][9].
• Use a guide RNA targeting a cut site close to the intended edit, and use a donor template matched to the edit size: ssODNs are commonly used for short sequence substitutions or small insertions, long ssDNA donors have been used for larger knock-ins in mouse embryos and primary human T cells, AAV donors have been paired with Cas9 RNP for gene addition, and plasmid or linear/double-cut donors have been used for larger cassette knock-ins[5][7][10][11][12][13].
• Use cell-culture medium and recovery conditions validated for the chosen cell type; small molecules that inhibit NHEJ or DNA-PK, such as Scr7, NU7441, KU-0060648, or DNA-PKcs-targeted approaches, have increased HDR in specific reported systems, but they should be treated as optional optimization variables rather than universal protocol components[14][15][16].
• Use PCR primers spanning the 5′ and 3′ knock-in junctions, primers or probes for the unedited allele, sequencing primers, and, when a fluorescent reporter is inserted, flow-cytometry or microscopy detection of the reporter as the primary enrichment or quantification readout[4][7][10][13].
• Use a sterile tissue-culture system, a delivery instrument appropriate to the biological system, such as electroporation or nucleofection for many cultured and primary cells or microinjection/electroporation for embryos, a thermocycler for genotyping PCR, a capillary or next-generation sequencer for allele confirmation, and flow cytometry or fluorescence microscopy when the donor encodes a reporter[4][6][8][10][11][13].
• Use a guide RNA targeting a cut site close to the intended edit, and use a donor template matched to the edit size: ssODNs are commonly used for short sequence substitutions or small insertions, long ssDNA donors have been used for larger knock-ins in mouse embryos and primary human T cells, AAV donors have been paired with Cas9 RNP for gene addition, and plasmid or linear/double-cut donors have been used for larger cassette knock-ins[5][7][10][11][12][13].
• Use cell-culture medium and recovery conditions validated for the chosen cell type; small molecules that inhibit NHEJ or DNA-PK, such as Scr7, NU7441, KU-0060648, or DNA-PKcs-targeted approaches, have increased HDR in specific reported systems, but they should be treated as optional optimization variables rather than universal protocol components[14][15][16].
• Use PCR primers spanning the 5′ and 3′ knock-in junctions, primers or probes for the unedited allele, sequencing primers, and, when a fluorescent reporter is inserted, flow-cytometry or microscopy detection of the reporter as the primary enrichment or quantification readout[4][7][10][13].
• Use a sterile tissue-culture system, a delivery instrument appropriate to the biological system, such as electroporation or nucleofection for many cultured and primary cells or microinjection/electroporation for embryos, a thermocycler for genotyping PCR, a capillary or next-generation sequencer for allele confirmation, and flow cytometry or fluorescence microscopy when the donor encodes a reporter[4][6][8][10][11][13].
Experimental Procedure
• Select a guide RNA whose Cas9 cut site is as close as possible to the intended edit, because reported HDR incorporation decreases as the edit is placed farther from the cut site; include silent blocking changes in the donor when they can disrupt the PAM or guide-recognition sequence without altering the intended biology, because this can prevent re-cutting of the corrected allele[5][12][17].
• Prepare the donor according to edit size and model system: use ssODN donors for short edits, long ssDNA donors for selected large insertions or mouse Easi-CRISPR designs, AAV donor vectors where AAV donor delivery is part of the validated system, or plasmid/double-cut donors where larger cassette insertion and donor release have been validated[7][10][11][13].
• For RNP editing, assemble Cas9 protein with sgRNA or crRNA:tracrRNA before delivery, and use chemically modified guide RNAs where supported for primary human T cells or CD34+ cells; for plasmid-based editing, prepare the Cas9/sgRNA expression construct and donor plasmid according to the cited mammalian-cell genome-engineering protocols[4][8][9][18].
• Seed or prepare cells so that they are healthy at delivery, and include an unedited control, a Cas9/guide-only control, a donor-only control when feasible, and a positive-control editing site if the cell type has not previously been edited in the laboratory[4][6][9][10].
• Deliver Cas9/guide components and donor template into the cells or embryos using the delivery method validated for the sample type; published examples include transfection of mammalian cells with Cas9/sgRNA plasmids and donor templates, electroporation or nucleofection of Cas9 RNP with donor DNA into human cells or primary immune cells, AAV donor delivery combined with Cas9 RNP, and zygote delivery of Cas9 RNP with long ssDNA donor[4][6][8][10][11][13].
• After delivery, culture cells under the recovery conditions used for the biological system, then collect bulk cells or embryos for genotyping, or isolate single-cell clones when a clonal knock-in line is required; reporter knock-ins may be enriched or quantified by fluorescence-based sorting or flow cytometry before molecular confirmation[4][7][10][13].
• For large cassette insertion, use 5′ and 3′ junction PCR to distinguish targeted integration from random donor integration or partial insertion, and sequence across both junctions and the edited locus; for short edits, amplify the target locus and use Sanger sequencing, restriction analysis if the edit creates/removes a restriction site, or amplicon deep sequencing to quantify HDR and indels[4][5][7][10].
• Calculate HDR efficiency as the percentage of alleles, cells, embryos, or clones carrying the intended donor-derived edit, and report NHEJ indel frequency, precise biallelic versus monoallelic editing when relevant, incomplete donor incorporation, and non-HDR donor insertion when the assay can detect these outcomes[5][7][10][13].
• Confirm edited clones by sequencing the complete edited region and both homology-arm junctions, and confirm loss or retention of blocking mutations as designed; for disease-model edits or functional knock-ins, validate expression or phenotype using downstream assays rather than relying only on PCR positivity[5][10][12].
• Use biological replicates for optimization experiments and compare conditions such as donor architecture, RNP versus plasmid delivery, guide choice, donor strand/asymmetry, and optional DNA-repair modulation only within the same cell type and locus, because HDR efficiency varies strongly across loci, cargos, and biological systems[6][7][12][14][15][16][17].
• Prepare the donor according to edit size and model system: use ssODN donors for short edits, long ssDNA donors for selected large insertions or mouse Easi-CRISPR designs, AAV donor vectors where AAV donor delivery is part of the validated system, or plasmid/double-cut donors where larger cassette insertion and donor release have been validated[7][10][11][13].
• For RNP editing, assemble Cas9 protein with sgRNA or crRNA:tracrRNA before delivery, and use chemically modified guide RNAs where supported for primary human T cells or CD34+ cells; for plasmid-based editing, prepare the Cas9/sgRNA expression construct and donor plasmid according to the cited mammalian-cell genome-engineering protocols[4][8][9][18].
• Seed or prepare cells so that they are healthy at delivery, and include an unedited control, a Cas9/guide-only control, a donor-only control when feasible, and a positive-control editing site if the cell type has not previously been edited in the laboratory[4][6][9][10].
• Deliver Cas9/guide components and donor template into the cells or embryos using the delivery method validated for the sample type; published examples include transfection of mammalian cells with Cas9/sgRNA plasmids and donor templates, electroporation or nucleofection of Cas9 RNP with donor DNA into human cells or primary immune cells, AAV donor delivery combined with Cas9 RNP, and zygote delivery of Cas9 RNP with long ssDNA donor[4][6][8][10][11][13].
• After delivery, culture cells under the recovery conditions used for the biological system, then collect bulk cells or embryos for genotyping, or isolate single-cell clones when a clonal knock-in line is required; reporter knock-ins may be enriched or quantified by fluorescence-based sorting or flow cytometry before molecular confirmation[4][7][10][13].
• For large cassette insertion, use 5′ and 3′ junction PCR to distinguish targeted integration from random donor integration or partial insertion, and sequence across both junctions and the edited locus; for short edits, amplify the target locus and use Sanger sequencing, restriction analysis if the edit creates/removes a restriction site, or amplicon deep sequencing to quantify HDR and indels[4][5][7][10].
• Calculate HDR efficiency as the percentage of alleles, cells, embryos, or clones carrying the intended donor-derived edit, and report NHEJ indel frequency, precise biallelic versus monoallelic editing when relevant, incomplete donor incorporation, and non-HDR donor insertion when the assay can detect these outcomes[5][7][10][13].
• Confirm edited clones by sequencing the complete edited region and both homology-arm junctions, and confirm loss or retention of blocking mutations as designed; for disease-model edits or functional knock-ins, validate expression or phenotype using downstream assays rather than relying only on PCR positivity[5][10][12].
• Use biological replicates for optimization experiments and compare conditions such as donor architecture, RNP versus plasmid delivery, guide choice, donor strand/asymmetry, and optional DNA-repair modulation only within the same cell type and locus, because HDR efficiency varies strongly across loci, cargos, and biological systems[6][7][12][14][15][16][17].
Troubleshooting
Problem: Low precise HDR but high indel formation.
• Possible cause: NHEJ is dominating repair after Cas9 cleavage.• Literature-supported solution: test reported HDR-enhancing or NHEJ/DNA-PK-inhibiting conditions as an optimization arm, and quantify both HDR and indels because pathway modulation is system-dependent[14][15][16].
Problem: The intended edit is underrepresented among edited alleles.
• Possible cause: the donor edit is too far from the Cas9 cut site.• Literature-supported solution: redesign the guide so the cut site is closer to the intended mutation, when target-site constraints permit[5][17].
Problem: Corrected alleles are re-cut, producing indels after HDR.
• Possible cause: the edited allele still contains an intact guide target or PAM.• Literature-supported solution: include donor-encoded silent blocking mutations in the PAM or guide-recognition sequence when biologically acceptable[5][17].
Problem: Apparent knock-in signal includes partial or non-HDR insertion.
• Possible cause: donor integration occurred outside the intended HDR structure.• Literature-supported solution: assay both 5′ and 3′ junctions and sequence the target locus rather than relying on reporter fluorescence or one-sided PCR alone[7][13].
References:
- [1]. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821. [Content Brief]
- [2]. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-823. [Content Brief]
- [3]. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826. [Content Brief]
- [4]. Ran FA, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308. [Content Brief]
- [5]. Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature. 2016;533(7601):125-129. [Content Brief]
- [6]. Lin S, et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife. 2014;3:e04766. [Content Brief]
- [7]. Zhang JP, Li XL, Li GH, Chen W, Arakaki C, Botimer GD, et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol. 2017;18(1):35. [Content Brief]
- [8]. Kim S, et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014;24(6):1012-1019. [Content Brief]
- [9]. Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015;33(9):985-989. [Content Brief]
- [10]. Schumann K, Lin S, Boyer E, Simeonov DR, Subramaniam M, Gate RE, et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci U S A. 2015;112(33):10437-10442. [Content Brief]
- [11]. Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, et al. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 2017;18(1):92. [Content Brief]
- [12]. Miura H, et al. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc. 2018;13(1):195-215. [Content Brief]
- [13]. Gaj T, et al. Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery. Nucleic Acids Res. 2017;45(11):e98. [Content Brief]
- [14]. Maruyama T, et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 2015;33(5):538-542. [Content Brief]
- [15]. Robert F, et al. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med. 2015;7:93. [Content Brief]
- [16]. Chu VT, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015;33(5):543-548. [Content Brief]
- [17]. Schubert MS, et al. Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair. Sci Rep. 2021;11(1):19482. [Content Brief]
- [18]. Richardson CD, et al. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol. 2016;34(3):339-344. [Content Brief]
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