Homology-directed repair FAQs

The sequence of the HDR template should match the intended sequence of the targeted genomic locus, including the desired edit flanked by 5' and 3' ends that share homology with the wild-type genomic locus. (These 5' and 3' sequences are referred to as "homology arms.")

Sequences corresponding to desired edits (insertions, substitutions, etc.) should be positioned in the middle of the HDR template. The exact sequence of the HDR template is independent of the position of the single guide RNA (sgRNA) or the cut site of the Cas9 nuclease, but efficient editing requires a template design in which the sequence(s) to be inserted are in relatively close proximity to the cut site of the Cas9-sgRNA ribonucleoprotein (RNP). (See FAQ below for more detail.)

The figure below demonstrates the design of HDR templates for engineering a single nucleotide substitution or a longer insertion, respectively, at corresponding genomic loci.

It has been demonstrated that the use of ssDNA results in lower toxicity and reduced frequencies of random integration relative to dsDNA (Roth et al. 2018; Li et al. 2017), benefits which can be especially important when working with difficult-to-engineer cell lines. For example, if you are seeking to fuse a fluorescent protein to an endogenous gene expressed in your target cells, the percentage of fluorescent cells in the overall edited population could be used as a proxy for the frequency of successful HDR. However, some subset of these fluorescent cells could have resulted from random integration of the HDR template in frame with a gene other than the intended genomic target. (Panel 4B in this poster includes data from our R&D team demonstrating this phenomenon.) Such events are less probable when ssDNA is used as a template for HDR.

References

Li, H et al., Design and specificity of long ssDNA donors for CRISPR-based knock-in. bioRxiv doi: https://doi.org/10.1101/178905 (2017).

Roth, T.L et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nat. Lett. 559, 405–409 (2018).

Single-stranded oligo deoxynucleotides (ssODNs) are produced routinely via chemical synthesis and can be used for engineering smaller edits (e.g., single nucleotide substitutions or shorter insertions <50 nt) as HDR templates with a total maximum length of 200 nt. For engineering larger insertions that necessitate HDR templates >500 nt, we recommend the use of ssDNA that can be produced with the Guide-it Long ssDNA Production System.

Currently, there is no clear rule regarding the preferential use of sense or antisense ssDNA for HDR templates longer than 200 nt. However, in the case of shorter single-stranded oligodeoxynucleotide (ssODN) templates, template polarity has been found to have an effect on efficiency (Bollen et al. 2018).

References

Bollen, Y., Post, J., Koo, B. & Snippert, H.J.G How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing. Nuc. Acid. Res. 46, 6435–6454 (2018).

It has been demonstrated that there is an exponential relationship between homology arm length and knockin efficiency, with ssDNA templates including homology arms 350–700 nt in length providing optimal performance (Li et al. 2017). In our own studies involving hiPSCs, optimal performance was observed with 350-nt homology arms, and the use of longer arms did not substantially increase knockin efficiencies.

It is important to note that using longer homology arms will increase the molecular weight of your HDR template, and since the amounts of HDR template used for electroporation are usually measured in µg, longer homology arms will correspond with fewer mols (copies) of template being introduced in the target cells, possibly impacting the overall percentage of HDR.

References

Li, H et al., Design and specificity of long ssDNA donors for CRISPR-based knock-in. bioRxiv doi: https://doi.org/10.1101/178905 (2017).

Cas9-sgRNA RNPs can re-cut a given genomic target following HDR if the editing outcome does not involve alteration of the PAM or sgRNA seeding region in the protospacer sequence, such that the RNP complex can still bind to the genomic target. Given this, a common strategy for increasing the percentage of successful HDR in coding regions is the incorporation of silent mutations in HDR templates that disrupt Cas9-sgRNA RNP binding without altering corresponding amino acid sequences.

If your PAM sequence is in a coding region and it can be silently mutated, it is advisable to do so to maximize the precision of genome editing and prevent subsequent Cas9 re-cutting and indel creation following HDR (Paquet et al. 2016). In seeking to mutate the PAM to disrupt RNP binding, please take into account other canonical PAM sequences (different from NGG) that can also be recognized less effectively by SpCas9 (i.e., NAG or NGA) (Zhang et al. 2014; Hsu et al. 2013) or Cas12a (i.e., TCTA, TCCA, or CCCA) (Yamano et al. 2017). Presented in the figure below are data demonstrating improved editing performance via incorporation of PAM mutations in HDR templates.

As mentioned above, the protospacer sequence can also be mutated. However, since some guide RNAs may tolerate a mismatch in the binding sequence, we recommend changing several bases as close to the PAM as possible in the seeding region of the sgRNA (Paquet et al. 2016; Kwart et al. 2017).

References

Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–32 (2013).

Kwart, D., Paquet, D., Teo, S. & Tessier-Lavigne, M. Precise and efficient scarless genome editing in stem cells using CORRECT. Nat. Protoc. 12, 329–354 (2017).

Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).

Yamano, T. et al. Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1. Mol. Cell 67, 633–645.e3 (2017).

Zhang, Y. et al. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci. Rep. 4, doi: 10.1038/srep05405 (2014).

It is highly recommended that the HDR insertion site is positioned within 10 nt upstream or downstream of the Cas9-sgRNA RNP cut site. For wild-type SpCas9, this is located 3–4 nucleotides upstream of the PAM. It has been demonstrated that there is an inverse relationship between HDR efficiency and the distance between the insertion site and Cas9-gRNA RNP cut site (Paquet et al. 2016).

References

Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).

It has been demonstrated that nucleosomes inhibit target cleavage by SpCas9 in vivo, suggesting that Cas9 activity is influenced by chromatin structure in addition to the inherent activity of a given sgRNA (Yarrington et al. 2018). Although DNA sequence is a significant determinant of site-specific indel profiles, it has been shown that packaging of DNA into chromatin may influence editing efficiency and the relative frequency of indels at a given locus (Chakrabarti et al. 2018). On the other hand, using imprinted genes as a test case, outcomes of gene editing events (i.e., the balance between HDR vs. NHEJ) were shown to be independent of chromatin state (Kallimasioti-Pazi et al. 2018).

References

Chakrabarti, A. M. et al. Target-Specific Precision of CRISPR-Mediated Genome Editing. Mol. Cell 73, 1–15 (2018).

Kallimasioti-Pazi, E. M. et al. Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair. PLOS Biol. 16, e2005595 (2018).

Yarrington, R. M., Verma, S., Schwartz, S., Trautman, J. K. & Carroll, D. Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo. Proc. Natl. Acad. Sci. 115, 9351–9358 (2018).

The development of novel methods for improving HDR efficiency is currently a major focus area in the genome editing field. Toward this end, the following approaches have shown promise:

• Fusion or attachment of Cas9-sgRNA RNP and template (Aird et al. 2018; Savic et al. 2018; Carlson-Stevermer et al. 2017)
• Restriction of Cas9 expression to S and G2 phases of the cell cycle, when the propensity for HDR is highest (Gutschner et al. 2016)
• Development of base editing for engineering single nucleotide substitutions (Rees and Liu 2018)
• Application of various chemical compounds that increase the percentage of HDR (Wienert et al. 2018; Riesenberg and Maricic 2018)

References

Aird, E. J., Lovendahl, K. N., St. Martin, A., Harris, R. S. & Gordon, W. R. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun. Biol. 1, 54 (2018).

Carlson-Stevermer, J. et al. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat. Commun. 8, 1711 (2017).

Gutschner, T., Haemmerle, M., Genovese, G., Draetta, G. F. & Chin, L. Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair. Cell Rep. 14, 1555–1566 (2016).

Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).

Riesenberg, S. & Maricic, T. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. Nat. Commun. 9, 2164 (2018).

Savic, N. et al. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. Elife 7, (2018).

Wienert, B. et al. Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair. bioRxiv 500462 (2018). doi:10.1101/500462

One of the most common genome editing outcomes is the introduction of an SNP in one allele and an indel in the other, such that it is especially challenging to obtain clones in which only one allele is modified (encoding for the desired edit) and the other one is unedited. To more efficiently obtain clones with SNP/wild-type heterozygosity, researchers have developed two strategies (Paquet et al. 2016):

• Use an sgRNA with a cut site farther away from the insertion site (>10 nt)

• Use an equal mix of mutant and wild-type HDR templates (one encoding for the SNP, the other encoding for the wild-type allele, both with the PAM sequence modified as explained in the previous FAQ)

Reference

Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).