Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair

Abstract

CRISPR-Cas proteins are RNA-guided nucleases 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 an exogenous DNA template during repair allows for the intentional, precise incorporation 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 (RNP) complexes. 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. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: https://www.idtdna.com/HDR.

Figure 1

Cas9 HDR strand preference and gRNA selection. (A) Schematic representation of targeting (T) and non-targeting (NT) donor template designs. The targeting strand is complementary to the gRNA sequence, whereas the non-targeting strand contains the guide and PAM sequence. (B) An EcoRI recognition site was inserted at a Cas9 cleavage site at 254 genomic loci in Jurkat and 239 genomic loci in HAP1 cells using either the T or NT strand as the donor template. RNP complexes (Alt-R S.p. Cas9 Nuclease complexed with Alt-R CRISPR–Cas9 crRNA and tracrRNA) were delivered at 4 µM along with 4 µM Alt-R Cas9 Electroporation Enhancer and 3 µM donor template by nucleofection. Total editing and perfect HDR was assessed via NGS. (C) Schematic of the gRNAs used to facilitate HDR insertion of an EcoRI site before the stop codon of GAPDH (TAA, red) in K562 cells using 13 guides around the desired HDR insertion location (blue, arrows indicate the 3’ end). The cleavage sites and associated distance to the desired insertion location (green) for each gRNA are indicated above the sequence shown. Both the T and NT strand were tested. (D) RNP complexes (Alt-R S.p. Cas9 Nuclease, Alt-R CRISPR–Cas9 crRNA and tracrRNA) for the 13 guides targeting GAPDH were delivered at 2 µM along with 2 µM Alt-R Cas9 Electroporation Enhancer and 2 µM donor template designed to insert an EcoRI site before the stop codon by nucleofection to K562 cells. HDR and total editing were assessed via NGS. Data are represented as means ± SEM of three biological replicates.

Figure 2

Cas9 D10A mediates efficient HDR distant from nick sites. (A) Ten HDR donor templates were designed with an EcoRI sequence positioned at varying distances (0-nt, 13-nt, 25-nt, 38-nt and 51-nt) from the left cleavage site of a paired-guide nickase design with a PAM-out orientation in HPRT1. ssODNs corresponding to the top and bottom (Btm) strand for each sequence were tested. Letters A–E indicate the position of the EcoRI insertion, the top strand ssODN is shown. (B) Cas9 D10A with gRNA pairs (left panel), or Cas9 WT with each of the individual gRNAs (middle and right panel) RNP complexes (Alt-R S.p. Cas9 D10A nickase or Alt-R S.p. Cas9 Nuclease complexed with Alt-R CRISPR–Cas9 crRNA and tracrRNA) were delivered at 4 µM (2 µM each RNP for nickase paired guides) along with 4 µM Alt-R Cas9 Electroporation Enhancer and 2 µM donor template by nucleofection to HEK293 cells (top) or K562 cells (bottom). HDR efficiency was evaluated by EcoRI cleavage of targeted amplicons. Data are represented as mean ± SEM of three biological replicates for D10A and two biological replicates for WT.

Figure 3

Blocking mutations improve HDR rates. (A) Schematic representation of a desired HDR-generated single base change (orange) 3’ of the PAM. In addition to the desired HDR mutation, a single blocking mutation in the seed region of the guide or PAM to prevent Cas9 re-cleavage was included in donor templates. Each position in the region indicated was changed to every possible alternate base in a unique donor template that also contained the desired HDR mutation. (B) HDR donors for two genomic loci were tested in HEK293 and K562 cells. In each case the donor contained an HDR mutation 3’ of the PAM, with or without a blocking mutation within the region indicated. HDR donors were delivered at 4 µM along with RNP complexes (Alt-R S.p. Cas9 Nuclease complexed with Alt-R CRISPR–Cas9 crRNA and tracrRNA) at 4 µM and with 4 µM Alt-R Cas9 Electroporation Enhancer by nucleofection. Each box represents the rate of perfect HDR including both the desired HDR mutation and blocking mutation (where applicable) as assessed by NGS. Blue indicates a higher HDR frequency, and red indicates a lower HDR frequency. (C) Blocking scores were calculated for 427 samples with known HDR frequencies and used to build a linear model (model = red line, standard error = blue highlight) to determine the optimum HDR efficiency. (D) Schematic representation of four unique HDR mutations that were designed using the Alt-R HDR Design tool either with or without the addition of silent mutations. (E) Four HDR mutations designed using the novel Alt-R HDR Design Tool with ( +) or without (−) silent mutations were tested in HEK293, Hela, and Jurkat cells. RNP complexes (Alt-R S.p. Cas9 Nuclease, Alt-R CRISPR–Cas9 crRNA and tracrRNA) were delivered at 4 µM along with 4 µM Alt-R Electroporation Enhancer and 4 µM donor template in HEK293 and Jurkat cells by nucleofection. RNP complexes (Alt-R S.p. Cas9 Nuclease complexed with Alt-R CRISPR–Cas9 sgRNA) were delivered at 2 µM along with 2 µM Alt-R Cas9 Electroporation Enhancer and 2 µM donor template in Hela cells by nucleofection. Perfect HDR rates were determined by NGS.

Figure 4

HDR mutation location determines donor strand preference. (A) Schematic representation of donor templates used to generate PAM-proximal and PAM-distal insertions 25 bases from a Cas9 cut site with no further mutations (None), PAM mutations (PAM), or mutations in the repair track with or without an additional PAM mutation. The NT strand ssODNs are shown. (B) Donor templates creating an EcoRI insertion 25 bases from the cut site at three genomic loci were delivered to Hela cells as the T or NT strand. Donor templates contained no further mutation (None), PAM mutation (PAM), or mutations in the repair track (Track). RNP complexes (Alt-R S.p. Cas9 Nuclease complexed with Alt-R CRISPR–Cas9 sgRNA) were delivered at 2 µM along with 2 µM Alt-R Cas9 Electroporation Enhancer and 0.5 µM donor template by nucleofection. Perfect HDR rates were determined by NGS. Data are represented as mean ± SEM of the three sites tested. (C) Schematic representation of donor templates used to generate PAM-proximal and PAM-distal insertions 20 bases from a Cas9 cut site with no further mutations (None), PAM mutations (PAM), or mutations in the repair track with or without additional PAM mutation. The NT strand ssODNs are shown. (D) Donor templates creating an EcoRI insertion at the cut site or 20 bases PAM-proximal or PAM-distal to the Cas9 cut site for 12 genomic loci were tested in Jurkat cells as the T or NT strand. RNP complexes (Alt-R S.p. Cas9 Nuclease complexed with Alt-R CRISPR–Cas9 sgRNA) were delivered at 4 µM along with 4 µM Alt-R Cas9 Electroporation Enhancer and 3 µM donor template by nucleofection. Perfect HDR rates were determined by NGS. Data are represented as mean ± SEM of the 12 sites tested.

Figure 5

Cas12a HDR gRNA selection and strand preference. (A) Schematic representation of targeting (T) and non-targeting (NT) donor template designs. The T strand is complementary to the gRNA sequence, whereas the NT strand contains the guide and PAM sequence. (B) HDR donors were designed with an EcoRI insert sequence positioned at varying distances from the from the first base of the Cas12a guide RNA ranging from 10 bases in the 5’ direction to 45 bases in the 3' direction for five genomic loci and delivered to HEK293 cells. RNP complexes (Alt-R A.s. Cas12a nuclease complexed with Alt-R CRISPR–Cas12a crRNA) were delivered at 5 µM along with 3 µM Alt-R Cpf1 Electroporation Enhancer and 3 µM donor template by nucleofection. HDR rates were assessed via EcoRI cleavage of targeted amplicons. The 21-base region where the gRNA targets is highlighted in green. The 4 base ‘TTTV’ PAM is highlighted in red. The gray shading indicates the confidence of fit. (C) An EcoRI restriction digest recognition site was inserted at position 16 of the gRNA sequence in 15 genomic loci in Jurkat and HAP1 cells using either the T or NT strand as the donor template and the combined results are graphed together. RNP complexes (Alt-R A.s. Cas12a Ultra nuclease complexed with Alt-R CRISPR–Cas12a crRNA) were delivered at 1 µM along with 3 µM Alt-R Cpf1 Electroporation Enhancer and 3 µM donor template by nucleofection. Total editing and perfect HDR was assessed via NGS. (D) Donors for two genomic loci were designed to insert an EcoRI site within the Cas12a guide sequence (position 15 of the guide) or outside of the guide sequence (24 bases from the start of the guide). ssODNs for these two insert locations were designed with blocking mutations in the PAM or guide sequence. The positions where blocking mutations were incorporated are indicated. (E) Donor templates for two genomic loci in HPRT1 were tested in Jurkat and Hela cells. RNP complexes (Alt-R A.s. Cas12a Ultra complexed with Alt-R CRISPR–Cas12a crRNA) were delivered at 2 µM along with 2 µM Alt-R Cpf1 Electroporation Enhancer and 3 µM donor template by nucleofection. HDR rates were assessed via NGS. Perfect HDR (blue), imperfect HDR (red) and total editing, which includes NHEJ events (black) are shown. Data are represented as mean ± SEM.