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Scientific ReportsMolecular BiologyPeer-reviewed journal article

Improved methods and optimized design for CRISPR Cas9 and Cas12a homology-directed repair

Mollie S. SchubertiD, Bernice Thommandru, Jessica WoodleyiD, Rolf TurkiD, Shuqi YaniD, Gavin KurganiD, Matthew McNeilliD, Garrett R. RettigiD

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Abstract

CRISPR-Cas proteins are used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing a DNA template during repair allows for precise introduction of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR-Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein complexes with synthetic guide RNAs. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. Additionally, we employ chemically modified HDR donor templates in combination with a small molecule to boost HDR efficiency up to 10-fold. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: www.idtdna.com/HDR

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (9 Apr 2021) · CC BY 4.0

Empirical ssODN design rules for Cas9, Cas9 D10A and Cas12a HDR, plus end-modified donors and an NHEJ-inhibiting small molecule, raise HDR up to 10-fold.

HDR is slower than error-prone NHEJ, and researchers are unsure how to choose guides, donor strand and blocking mutations; Cas12a had not been studied systematically. This work derives empirical rules for Cas9, D10A nickase and Cas12a and builds the Cas9 and D10A rules into an online donor design tool.

Key results

  1. ShownFor WT Cas9, the best ssODN strand depends on where the edit sits: the non-targeting strand for PAM-distal inserts, the targeting strand for PAM-proximal inserts. Repair-track mutations help PAM-distal edits most.

    HeLa, 3 loci: PAM-distal NT 12.7% vs T 1.6% HDR; PAM-proximal T 8.6% vs NT 0.8%. Repair-track mutations gave 3.4-fold over PAM mutation alone for PAM-distal inserts (p <0.01, paired t-test). The strand preference held at all 12 loci tested (inserts 20 bases from the cut). · HDR mutation location determines donor strand…

  2. ShownAlt-R end-modified donors and the HDR Enhancer compounds each raise HDR, and the combination gives the highest frequencies in the tested loci.

    HeLa, 8 loci: PS-modified 3.1-fold and Alt-R modified 5.2-fold over unmodified (1.7-fold over PS). HPRT1 5.9% unmodified, 28.4% Alt-R, 52.0% with Enhancer V1; SAA1 4.1%, 18.5%, 39.4% (9.6-fold). Enhancer V2 reached 3.2-fold in Jurkat (Cas9). · Alt-R modified HDR Donor oligos and Alt-R HDR…

  3. ShownBlocking mutations in the PAM or seed raise Cas9 HDR; two are enough, and extra PAM mutations hurt when the insert lies in the guide seed or PAM. A PSSM-based blocking score was built into the design tool.

    One PAM blocking mutation (2nd or 3rd base) lifted HDR from <2% (control) to 8.0-17.8%; 3 or 4 blocks were no better than the best 2. Score model used 374 designs from 9 guides and 3 cell lines in the text (the legend says 427 samples); scores <1.97 predicted a positive effect. · Optimizing placement and number of blocking…

  4. ShownPaired Cas9 D10A nickase RNPs in PAM-out orientation support HDR across the span between the nicks, where WT Cas9 with either guide declines with distance, and show no donor strand preference.

    HPRT1, HEK293: D10A HDR 13-25% across the 51-nt region. Optimal nick spacing 40-68 nt (D10A editing 86.3% at 68 nt vs 29.8% at 85 nt). Replicated at AAVS1 (46-nt spacing) with D10A above WT at the centered position. · Cas9 D10A mediates efficient HDR distant from…

  5. ShownCas12a HDR peaks for inserts at guide positions 12-16 and favors the non-targeting strand donor. Blocking mutations restore HDR for inserts placed outside the guide.

    15 guides in Jurkat and HAP1: T strand gave lower total editing and HDR (significant NT preference). Insert outside the guide: HDR 0.7% without a block vs 13.3% with a PAM mutation and 13.0% with a position 14 mutation (2 HPRT1 loci). · Optimized design rules for HDR with Cas12a

Methods, data and reproducibility

ssODN donors carrying EcoRI insertions or single-base changes were tested with Cas9, D10A, H840A and Cas12a RNPs in mammalian cell lines. Total editing and perfect HDR were scored by amplicon NGS or EcoRI digestion, and the resulting rules were encoded in a web tool.

  • EcoRI insertion at Cas9 cut sites, T vs NT donor strand, NGS readout (Jurkat and HAP1) · human (cell lines; GRCh38 alignment) · n = 254 loci (Jurkat); 239 loci (HAP1) · PRJNA638623
  • GAPDH (13 guides, K562 and HEK293) and TNPO3 (HEK293) guide selection vs distance to insertion · human (cell lines) · n = 13 guides at GAPDH · PRJNA638623
  • Paired nickase (D10A, H840A) vs WT Cas9 HDR at HPRT1 and AAVS1 in HEK293 and K562 · human (cell lines) · n = AAVS1 46-nt PAM-out design; HPRT1 51-nt site
  • Blocking mutation scans and score model (HEK293, K562, Jurkat, Hepa1-6) · mammalian cell lines (HEK293, K562, Jurkat, Hepa1-6) · n = 374 designs (text) or 427 samples (legend); 9 guides, 3 cell lines · PRJNA638623
  • Repair-track mutation and strand experiments with EcoRI inserts at suboptimal distance (HeLa, Jurkat) · human (cell lines) · n = 3 loci (HeLa); 12 loci (Jurkat and HeLa) · PRJNA638623
  • Cas12a insertion position scan at HPRT1 (HEK293) and strand test (Jurkat, HAP1) · human (cell lines) · n = 5 HPRT1 sites; 15 loci · PRJNA638623
  • Modified donor oligo comparison (unmodified, PS, Alt-R) in HeLa · human (cell line) · n = 8 loci; 3 loci with Enhancer V1 · PRJNA638623
  • HDR Enhancer V1 and V2 tests with Cas9 and Cas12a (HEK293-Cas9, K562, Jurkat) · human (cell lines) · n = 4 Cas9 loci; 4 Cas12a loci · PRJNA638623
Methods & tools
Alt-R HDR Design Tool, Alt-R S.p. Cas9, HiFi Cas9, D10A, H840A nucleases, Alt-R A.s. Cas12a V3 and Ultra, Alt-R HDR Donor Oligos, Alt-R HDR Enhancer V1 and V2, T7 Endonuclease I assay, EcoRI digestion assay, Illumina MiSeq amplicon sequencing, Picard tools v2.9, flash v1.2.11, minimap2 v2.12, GraphPad Prism 8
Software & environment
Picard tools v2.9, flash v1.2.11, minimap2 v2.12, GRCh38 reference, GraphPad Prism 8; MiSeq paired-end 150 bp reads with V2 chemistry.
Reproducibility gaps
The custom NGS analysis pipeline has no stated code link. Alt-R donor end-block chemistry is proprietary, and NGS data are given only as a BioProject accession (PRJNA638623).
Limitations (5)
  • Cas12a PAM-proximal insertions and their strand preference were not thoroughly investigated, so it is unclear whether the targeting strand helps there as it does for Cas9. The authors say further experimentation is required. · stated by the authors
  • Paired nickase designs need two guides with suitable spacing, activity and orientation, which rules this strategy out at some sites. · stated by the authors
  • The lack of base preference in blocking mutations could reflect a small or site-specific effect; a larger dataset might resolve differences. Cas12a blocking-mutation rules are still being worked out. · stated by the authors
  • Several experiments rest on few loci or replicates (WT Cas9 arm used two biological replicates) and only immortalized cell lines; no primary or in vivo cells were tested. · noted in this summary
  • Key comparisons (20+ other donor modifications, homology arm effects, Enhancer V2 combinations) are 'data not shown'; the statistics section states unpaired t-tests while results cite paired tests. · noted in this summary

Read this if you design ssODN knock-ins with Cas9, Cas9 D10A or Cas12a and want empirical rules on guide choice, donor strand, blocking mutations and donor chemistry.

Citations
123
FWCI
5.49
Field percentile
top 2%

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