Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

Background While CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing. Results We systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform precise gene targeting efficiently across multiple genomic loci using single-stranded oligodeoxynucleotide (ssODN) donor templates with homology arms as short as 17 nucleotides. Strikingly, the two Cpf1 nucleases exhibit a preference for ssODNs of the non-target strand sequence, while the popular Cas9 enzyme from Streptococcus pyogenes (SpCas9) exhibits a preference for ssODNs of the target strand sequence instead. Additionally, we find that the HDR efficiencies of Cpf1 and SpCas9 can be further improved by using asymmetric donors with longer arms 5′ of the desired DNA changes. Conclusions Our work delineates design parameters for each CRISPR-Cas system and will serve as a useful reference for future genome engineering studies. Electronic supplementary material The online version of this article (10.1186/s13059-018-1445-x) contains supplementary material, which is available to authorized users.


Figure S1
Confirmation of cleavage activities of our CRISPR constructs.
We cloned each Cas endonuclease and its cognate sgRNA into the same plasmid backbone and then tested the new constructs for cleavage activities. As determined by T7E1 assays, we observed robust editing activities for all the five nucleases, namely SpCas9, SaCas9, NmCas9, AsCpf1, and LbCpf1. The targeted sites and the primers used for the assays are given in Tables S1, S2 respectively. Data represent mean ± s.e.m (n ≥ 3 per construct). There was no significant difference in indel rates between CAG-expressed enzymes and EF1αexpressed enzymes at all the genomic loci tested. (N.S.: not significant; Student's t-test) b H3K27ac ChIP-seq data for the five selected genes. Red boxes indicate the start of each gene. We observed larger ChIP-seq peaks for genes that were more highly expressed. e Boxplot summarizing the rates of indel formation quantified by T7E1 assays (blue) or deep sequencing experiments (red). P-values were calculated using the Wilcoxon rank sum test.

Figure S3
Extent of genome modifications as determined by T7E1 assays.
To ensure a fair comparison, matched target sites flanked by optimal PAMs for different Cas endonucleases were selected. Spacer lengths from 17nt to 23nt inclusive were tested. The cells were harvested 24 hours after transfection. Data represent mean ± s.e.m (n ≥ 6 per target site). (N.D.: not detected) ae Intronic sites were selected in genes with varying expression levels. The genes were a ALK, b EGFR, c NF1, d KDM6A, and e STAG2. We noted that SaCas9 and NmCas9 required incompatible PAMs and hence had to be evaluated separately. The left panels (group A) contained genomic loci targeted by SpCas9, SaCas9, AsCpf1, and LbCpf1. The right panels (group B) contained genomic loci targeted by SpCas9, NmCas9, AsCpf1, and LbCpf1.
fh Additional target sites in protein-coding regions were selected in f APC, g ATM, and h KDM5C (left panel,18nt) and ALK (right panel,19nt). The same genome loci as those in Figure S3 were targeted, with the rate of indel formation measured by deep sequencing instead. The cells were harvested 24 hours after transfection.

Figure S5
Evaluation of various CRISPR-Cas systems in NHEJ-mediated genome editing using matched spacers or matched seeds. a, b Summary of matched target site activities (see Figure S3) for SpCas9, either a SaCas9 or b NmCas9, AsCpf1, and LbCpf1 based on T7E1 assays. Each horizontal bar indicates the mean of the editing activities for the indicated enzyme and range of spacer lengths. c, d Extent of genome modifications at a target locus in the c CACNA1D or d PPP1R12C gene whereby the Cas9 and Cpf1 nucleases had overlapping seed regions. Three different spacer lengths (17nt, 20nt, and 23nt) were tested. The cells were harvested 24 hours after transfection and the editing efficiencies were determined by T7E1 assays. Data represent mean ± s.e.m (n ≥ 6).

Figure S6
We observed in HEK293T cells that SpCas9 was the only Cas nuclease that exhibited robust editing activity with short 17nt spacers. To confirm the results, we targeted the B1 (17nt) site located within the ALK gene in other cell lines, namely HCT116 and PC9.
All cells were harvested at 24 hours after transfection. From T7E1 cleavage assays, we again found that only SpCas9 was able to modify the genome robustly at this genomic locus, thereby verifying our observation in the HEK293T cell line. (N.D.: not detected)

Figure S7
Editing activities of NmCas9 and Cpf1 nucleases at target sites of lengths 24-25nt.
a Nine additional target sites (group C) were selected in six different highly expressed genes (WDR5, COPA, STAG2, HDAC2, GLUL, and PARK7) based on RNA-seq experiments.
b H3K27ac ChIP-seq data for the COPA and PARK7 genes. The ChIP-seq peaks for STAG2 are shown in Figure S2b, while the peaks for the remaining genes (WDR5, HDAC2, and GLUL) are provided in Figure S9c.
c Bar graph showing the editing activity of NmCas9 and the two Cpf1 nucleases at the nine newly selected target sites, which take the form TTTN-N24-25-NNNNGATT (see Table S5).
The cells were harvested 24 hours after transfection and then the editing frequencies were quantified by T7E1 assays. Data represent mean ± s.e.m (n ≥ 5). (N.D.: not detected) d Strip chart summarizing the editing efficiencies of NmCas9, AsCpf1, and LbCpf1 at perfectly matched target sites of longer lengths (24-25nt).

Figure S8
Confirming the low editing activity of NmCas9 in multiple human cell lines.
We found that NmCas9 failed to edit the HEK293T genome at many of the tested sites, while other nucleases were able to generate indels. To confirm the results, we targeted three distinct sites in various alternative cell lines, namely a B5 (21nt) in the ALK gene, b C4 (24nt) in the STAG2 gene, and c C1 (24nt) in the WDR5 gene. From T7E1 assays, we observed that NmCas9 again produced weaker cleavage bands than the other Cas enzymes at the three sites in all the additional cell lines tested, thereby verifying our earlier results in HEK293T cells. a To better characterize the editing activity of SaCas9, we selected six new target sites in four lowly expressed genes (HNF4A, ADARB2, ASCL2, and KCNA1) (light blue bars) as well as 12 new target sites in six highly expressed genes (WDR5, HDAC2, GLUL, SRSF1, SOD1, and VIM) (dark blue bars). The expression levels of all the nine genes were obtained from RNA-seq experiments. Data represent mean ± s.e.m (n = 6).
b H3K27ac ChIP-seq data for the four lowly expressed genes. No obvious peak could be seen in the plots.
c H3K27ac ChIP-seq data for the six highly expressed genes. We observed clear ChIP-seq peaks for all these genes.

Figure S10
Editing efficiencies at group D target sites.
The activity of SpCas9 and SaCas9 was measured either by a the T7E1 cleavage assay or by b Illumina deep sequencing experiments 24 hours post-transfection. D1-D6 are in lowly expressed genes, while D7-D18 are in highly expressed genes (see Table S6). Data represent mean ± s.e.m (n ≥ 4). (N.D.: not detected)

Figure S11
Relationship of DNA cleavage efficiency with gene expression and target specificity.
a Impact of gene expression on editing efficiency. We divided the target sites into those that occur in lowly expressed genes (FPKM < 25, blue boxplots) and those that occur in highly expressed genes (FPKM ≥ 25, red bloxplots) using our RNA-seq data. The FPKM value of 25 was chosen to divide the target sites into two groups of roughly equal sizes for the five Cas nucleases. Here, all sgRNAs were considered in the analysis. Overall, we found from our T7E1 assays that SpCas9, AsCpf1, and LbCpf1 were able to edit highly expressed genes more efficiently than lowly expressed genes (P < 0.05, Wilcoxon rank sum test). In contrast, the performance of SaCas9 was not influenced by gene expression. For NmCas9, its activity appeared to be dependent on gene expression as well, but this may be due to the numerous zero values resulting from non-optimal sgRNAs.
b Similar analysis to a, except that only sgRNAs of optimal lengths were considered. In the current study, we set the optimal lengths of SpCas9 as 17-22nt inclusive, SaCas9 as ≥ 21nt, NmCas9 as ≥ 19nt,AsCpf1 as ≥ 19nt,and LbCpf1 as ≥ 19nt. Again, the activity of SpCas9, AsCpf1, and LbCpf1 showed a significant dependence on gene expression (P < 0.05, Wilcoxon rank sum test). In contrast, the performance of the two smaller Cas enzymes, SaCas9 and NmCas9, was less affected by expression levels of the targeted genes.
c Comparison of AsCpf1 with either SpCas9 (left boxplot) or LbCpf1 (right boxplot). Only sgRNAs of the optimal lengths for SpCas9 and the Cpf1 nucleases (19-22nt inclusive) were considered. From T7E1 assays, we found that the editing activity of AsCpf1 was significantly lower than both SpCas9 and LbCpf1 (P < 0.05, Wilcoxon rank sum test).
d To assess the specificities of SpCas9, AsCpf1, and LbCpf1, we examined the tolerance of these enzymes to single mismatches along the spacer targeting the A17 site in the NF1 gene.
Red letters indicate the mutated bases.
e Using the spacers indicated in d, we determined the editing activities of SpCas9, AsCpf1, and LbCpf1 by T7E1 assays. The cells were harvested 24 hours after transfection. For all three nucleases, we observed an increased tolerance to mismatches around the middle of the spacer. Importantly, while SpCas9 and LbCpf1 exhibited higher cleavage efficiencies than AsCpf1 with a perfect matched (PM) spacer, they also showed an overall higher tolerance to mismatches between the spacer and the target DNA. Data represent mean ± s.e.m (n ≥ 5).

Figure S12
Identification of Cas enzymes with the highest cleavage efficiencies at different spacer lengths. At each target site, we asked which of the four nucleases exhibited the highest editing activity. We then determined the total number of sites that each nuclease emerged as the best performing enzyme.
a Percentages of the set of Group A target sites (of the specified lengths), including those in the CACNA1D gene, whereby each indicated enzyme generated the largest amount of indels.
SpCas9 was the best performing nuclease for spacers that were 17-20nt long, while SaCas9 and LbCpf1 were the best performing nucleases for spacers that were 21-23nt long.
b Percentages of the set of Group B target sites (of the specified lengths), including those in the PPP1R12C gene, whereby each indicated enzyme generated the largest amount of indels.
SpCas9 was again the best performing nuclease for 17-20nt long spacers, while LbCpf1 remained the best performing nuclease for 21-23nt long spacers. Notably, there was no target site of any length whereby NmCas9 exhibited the highest cleavage efficiency.

Figure S16 Correct versus incorrect integrations of restriction sites.
We utilized our deep sequencing data to investigate the rate of erroneous incorporations of XbaI into the a CACNA1D or b PPP1R12C genomic locus. As a baseline, we also determined the extent of XbaI integrations in NHEJ-mediated editing experiments when no donor template was provided. Blue diamond data points indicate SpCas9, gray triangle data points indicate AsCpf1, and orange triangle data points indicate LbCpf1. b Extent of incorporating the HindIII recognition sequence into the A3, A11, or B8 target sites. Donor ssODNs with 27nt homology arm lengths were used. The cells were harvested for RFLP analysis 72hr post-transfection. The Cpf1 nucleases consistently gave more digested products than SpCas9. Data represent mean ± s.e.m (n ≥ 6). (** P < 0.01, *** P < 0.001; Student's t-test) c Extent of precise gene editing by SpCas9, AsCpf1, and LbCpf1 at the B8 locus when ssODNs of different homology arm lengths (17-27nt inclusive) were used. The cells were harvested for RFLP analysis 72hr post-transfection. Data represent mean ± s.e.m (n ≥ 2). (* P < 0.05, ** P < 0.01, *** P < 0.001; Student's t-test)

Figure S19 Correct versus incorrect integrations of restriction sites.
We utilized our deep sequencing data to investigate the rate of erroneous restriction site e B8 (in EGFR), or f B18 (in STAG2) target locus. As a baseline, we also determined the extent of restriction site integrations in NHEJ-mediated editing experiments when no donor template was provided. Blue diamond data points indicate SpCas9, red diamond data points indicate either SaCas9 or NmCas9, gray triangle data points indicate AsCpf1, and orange triangle data points indicate LbCpf1. Representative Integrative Genomics Viewer (IGV) screenshots of some of the sequencing reads covering target sites located in the a CACNA1D, b PPP1R12C, c ALK (A3), d ALK (B4), e EGFR (A11), f EGFR (A12), g EGFR (B8), and h STAG2 (B18) genes. Asterisks indicate the reads where the relevant restriction site was correctly incorporated into the targeted locus using ssODNs as donors. We could visually observe that SpCas9 was able to produce as many random indels as the Cpf1 nucleases, but smaller fractions of the reads for SpCas9 contained the desired genome modifications. We performed PCR to check whether the P2A-eGFP cassette was correctly integrated at the C-terminal end of a CLTA and b GLUL. Red arrows indicate the primers for the junction PCR, while blue arrows indicate the primers for the control PCR. In the absence of a sgRNA, no band was observed in the junction PCR. However, in the presence of the relevant sgRNA, bands of the expected size were observed for all the Cas nucleases, indicating that the cassette was targeted to the right genomic loci.

Figure S29
Pairwise comparison of SpCas9 and LbCpf1.
We compared the ability of SpCas9 and LbCpf1 to edit genes of different expression levels.
Only sgRNAs of the optimal lengths for both SpCas9 and LbCpf1 (19-22nt inclusive) were considered. Overall, from a deep sequencing analysis and b T7E1 assays, we found that the editing activities of SpCas9 and LbCpf1 were not significantly different in both lowly expressed and highly expressed genes (P > 0.05, Wilcoxon rank sum test).       Table S7 PCR primers used in T7E1 assays. A1_ALK_T7E1_FOR  AAA TCT CAT GGG TGC AGA GG  A1_ALK_T7E1_REV  CCA CGG TAA AAA GGC CAT AA  A2_ALK_T7E1_FOR  TGG TTG CTC AGG AAG ATG AA  A2_ALK_T7E1_REV  ACA CGT GAA GGC ATT TTT CC  A3_ALK_T7E1_FOR  TCG TCC TGT TCA GAG CAC AC  A3_ALK_T7E1_REV  TGT GTC CCT GGC AAA TAT CA  A4_ALK_T7E1_FOR  ACA GAG GGT TCA CGT TCT CG  A4_ALK_T7E1_REV GTT CCA GGC ATT CCT TCT GA A5_ALK_T7E1_FOR

Primer Name Primer Sequence
CCA TGC ATG ATT TGG GTA GA A5_ALK_T7E1_REV AGC ACT TTG GCA GAA AGG AA A6_ALK_T7E1_FOR CCC CAG CTT TCA CAT CAT CT A6_ALK_T7E1_REV GTG TGT GCA TGG TGT GTG AC A7_ALK_T7E1_FOR CCT GCC ATT CTT CCA CTG AT A7_ALK_T7E1_REV GTG TAG CCG ATC CAA CCA TT  B1_ALK_T7E1_FOR  AGC AGG GGC TGG ATT TAT TT  B1_ALK_T7E1_REV  CTC AGC CTA AAG CCC TGT GT  B2_ALK_T7E1_FOR  CTT TCA AAG GTG TGG GGA AG  B2_ALK_T7E1_REV  GCA GAT GGC TGT CTT CTG GT  B3_ALK_T7E1_FOR  AAC TGG CTA CAG CCC AAG AA  B3_ALK_T7E1_REV  GCC AGT GGA CAA TTG ATG TG  B4_ALK_T7E1_FOR  TTG TTG TTG GGA CGT GTC AT  B4_ALK_T7E1_REV  AGA TAC TGG GCA GCA AAT GG  B5_ALK_T7E1_FOR  GGG GCT TCG TTT CTT ATT CC  B5_ALK_T7E1_REV  TTT CTG TCC AGC TCC CAA GT  B6_ALK_T7E1_FOR  AAA GTC ACA CCC CAT TCT GC  B6_ALK_T7E1_REV  CTG TGT CTT CCA GGA TGC AA  CDS_ALK_19nt_T7E1_FOR  GAAAGCCCAAGGTGTGAAGA  CDS_ALK_19nt_T7E1_REV  TGAGCCTCTGCTTTGTCAGA  A8_EGFR_T7E1_FOR  CTA TGG TTG CCC AAA AGC AT  A8_EGFR_T7E1_REV  GCC TGG AGA AAG ATG GAC AA  A9_EGFR_T7E1_FOR  TTG GCT TCC TAG ATC CCT GA  A9_EGFR_T7E1_REV  GGC ACA CAC GTG CAG ATA AG  A10_EGFR_T7E1_FOR  TGG TGA CTG TGT GAG CGA AT  A10_EGFR_T7E1_REV  TGC TTT ACG AGG CCA ATT TC  A11_EGFR_T7E1_FOR  CCC TGC CAC TCA TCA AAA AT  A11_EGFR_T7E1_REV  GGA GAG AAA TGC TCC TGC AC  A12_EGFR_T7E1_FOR  CAG TGA GTC ACA CCC TGG AA  A12_EGFR_T7E1_REV  GTG GAG GAG GAG ATG GGA AT  A13_EGFR_T7E1_FOR  TAA AAA CCT GGC CCA GAA CA  A13_EGFR_T7E1_REV  ATA GCA TGG CTG CTG CAT AA  A14_EGFR_T7E1_FOR  TGA GCC CCA TTT TGA AAC AC  A14_EGFR_T7E1_REV  AGG GAA GCT GAG GAA GGA AC  B7_EGFR_T7E1_FOR  TTT TGG TTC CTC CAT CTT TGA  B7_EGFR_T7E1_REV  TCC AGA GTG CCC ATG TCT TAC  B8_EGFR_T7E1_FOR  GGA GCA TGA AGC AGT CAT CA  B8_EGFR_T7E1_REV  CGA AGG ACT TCG ATT TTG CT  B9_EGFR_T7E1_FOR  CAG CTT TGG GAC AAG GAG AG  B9_EGFR_T7E1_REV  CGC CTC TCA CTC TGA ACT CC  B10_EGFR_T7E1_FOR  ATT GTT GGC TGT TCG GTG TT  B10_EGFR_T7E1_REV ATG  T  B13_NF1_T7E1_REV  GTG TCA ATC AAG GCA TCA AGA A  B14_NF1_T7E1_FOR  TGA ATG CCC AAT TCC TTT TC  B14_NF1_T7E1_REV  GCA TGG AGT CTG CCA ATT CT  A22_KDM6A_T7E1_FOR  TTA ACA CAC ACA AAA AGG ATG GA  A22_KDM6A_T7E1_REV  TTT GAG ACG TAG TTT TGC TGT CA  A23_KDM6A_T7E1_FOR  CCT GCA AAT AAC AAG GGG TCT  A23_KDM6A_T7E1_REV  GTC TGG GGC TAA TCA AAG CA  A24_KDM6A_T7E1_FOR  CTT CCC CTG CTC TCA AGA AG  A24_KDM6A_T7E1_REV  TAT AGG GTG GTG GGG ATT GA  A25_KDM6A_T7E1_FOR  CCC TCT TCC CCC TAC TTC AT  A25_KDM6A_T7E1_REV  ATA GCT GCA CAA GCG GAA GT  A26_KDM6A_T7E1_FOR  GAG ACG GTC TTC ATA TTT TCC AA  A26_KDM6A_T7E1_REV  TGT GGC TAT GAT GTT TCG AAC T  A27_KDM6A_T7E1_FOR  GAT GAG CCT TGG ATA AAA CCA G  A27_KDM6A_T7E1_REV  CAC AAT CTG AAA ATC CAT GAG G  A28_KDM6A_T7E1_FOR  CCG ACC TAA ACT CCG TGA AA  A28_KDM6A_T7E1_REV  CCT CTT TGG GTT CGT GAG AT  B15_KDM6A_T7E1_FOR  ACT ACC TGC ACC CTG CAC TT  B15_KDM6A_T7E1_REV  GTC TTG CTG GGC TTT TTC TG  B16_KDM6A_T7E1_FOR  GCA CAG AAG AAA CAG ACT GAA AAG  B16_KDM6A_T7E1_REV  ACA TCT TTT CAC ATC ACA TGG ACT  B17_KDM6A_T7E1_FOR GGA  CACNA1D_T7E1_REV  TGG AGT TTC TGC TCC CAT TT  PPP1R12C_T7E1_FOR  AAG TTC TGT GGG AGG GGA CT  PPP1R12C_T7E1_REV  TCT CAG TTC TCG CAC TGC TG  CLTA_T7E1_FOR  AGG CAG TTG CTT GTG TAG CA  CLTA_T7E1_REV  TTA GTT CAA GGC AGG GCT GT  GLUL_T7E1_FOR  GGC TCC ATA CCT GGA GAC AA  GLUL_T7E1_REV  CCT CTA TCC CAG CCA AAC AA  C1_WDR5_T7E1_FOR  GTA CTC GGC CCT AGA TGC AG  C1_WDR5_T7E1_REV  CTG CAG TTC AAT CGG TTT CA  C2_COPA_T7E1_FOR  CCA CAG CAG CTT TCT TTC CT  C2_COPA_T7E1_REV  GCA ATC CTC TGC CTC AGC  C3_STAG2_T7E1_FOR  ATT TAT GCA GGC CAC CAC TC  C3_STAG2_T7E1_REV  GGG ACC ACA TTC ATT GCC TA  C4_STAG2_T7E1_FOR  GCC CCT CAG TTG TAA CAT TCA  C4_STAG2_T7E1_REV TCA GCA ACC GTG TGA GAA AG C5_HDAC2_T7E1_FOR GGT AGT GAT GGG CTC TGA GG C5_HDAC2_T7E1_REV TTC CCA ATC CAA TCC ATG TT C6_HDAC2_T7E1_FOR CCA ATT CCA TTA AGA CCA GCA C6_HDAC2_T7E1_REV GGT GGC TCT GTT CTC TGT CC C7_GLUL_T7E1_FOR ACT CAG GGG AGC AAA GGA AG C7_GLUL_T7E1_REV TCT GCT CTT GGA GGA GAT GG C8_GLUL_T7E1_FOR GGC GTG GTC CTA GTT TAT GC  C8_GLUL_T7E1_REV  CCC TAG ACA ACA CCC ATC CA  C9_PARK7_T7E1_FOR  TCA CTA TGT TGC CCA AGC AG  C9_PARK7_T7E1_REV  TGC CAT TTA GTG GCT GTC TG  D1_HNF4A_T7E1_FOR  ATG CAC CTT GTT CCT TTC AAC T  D1_HNF4A_T7E1_REV  CCA ACA ATG GCT TCA TTC AGT A  D2_HNF4A_T7E1_FOR  GCT GGT AGA GCA GGT GAG ATG  D2_HNF4A_T7E1_REV  AGT GCC TGG GAG TAA GGA AGA  D3_ADARB2_T7E1_FOR  CAA GAA GAA GGC CAA GAT GC  D3_ADARB2_T7E1_REV  GCA CCT GTT CTC CCA TCA AT  D4_ASCL2_T7E1_FOR  CTC CCC ACA GCT TCT CGA C  D4_ASCL2_T7E1_REV  GGC TGC ACT CCA GAT CTC A  D5_D6_KCNA1_T7E1_FOR  GTC TCC GTC ATG GTC ATC CT  D5_D6_KCNA1_T7E1_REV  AGG GCA ATT GTT AGC ACA CC  D7_WDR5_T7E1_FOR  TTG CTG GTG ACA TTT CTT GC  D7_WDR5_T7E1_REV  AGC CTC AGC ACC TCC TGT C  D8_WDR5_T7E1_FOR  TGT GAA TGG TTG TGG CAA GT  D8_WDR5_T7E1_REV  TAA CTG CTG TGC AGG TGA GC  D9_HDAC2_T7E1_FOR  GCT CAA AAA TGG GTT TCC TG  D9_HDAC2_T7E1_REV  GAG AAG GGC TTC ATG CTT TG  D10_HDAC2_T7E1_FOR  AAT TCT ACC ACC TTG CCC TCT  D10_HDAC2_T7E1_REV  AGA ATC AAT GTG GGC CTG AC  D11_D12_GLUL_T7E1_FOR  TGG GAG CAG ACA GAG CCT AT  D11_D12_GLUL_T7E1_REV  TTG CAA GTC ATC CTG CAA AG  D13_SRSF1_T7E1_FOR  CAA CTG AGC GAG CTT CTC CT  D13_SRSF1_T7E1_REV  GGT AAA TCA CCA CAG CAG CA  D14_SRSF1_T7E1_FOR  ATG TTT ACC GAG ATG GCA CTG  D14_SRSF1_T7E1_REV  TCA AAG ACA CGA AGG GAA TGT  D15_SOD1_T7E1_FOR  TTG CCA ATT TCG CGT ACT G  D15_SOD1_T7E1_REV  ACC CGC TCC TAG CAA AGG T  D16_SOD1_T7E1_FOR  TTG GGT ATT GTT GGG AGG A  D16_SOD1_T7E1_REV  TCA CAG GCT TGA ATG ACA AAG  D17_VIM_T7E1_FOR  CCT CCT ACC GCA GGA TGT T  D17_VIM_T7E1_REV  GGC TTT GTC GTT GGT TAG C  D18_VIM_T7E1_FOR  GCA TAA GCC ACC ATG ACC A  D18_VIM_T7E1_REV TCT TGG CAG CCA CAC TTT C  A1_ALK_Adapter_FOR  GCG TTA TCG AGG TCT TCT GAA AGA TGC ACT  CAA GAT GT  A1_ALK_Adapter_REV  GTG CTC TTC CGA TCT ATA AAA CAC CTT GGG  GAA AAC A  A2_ALK_Adapter_FOR  GCG TTA TCG AGG TCT AGG CCC GTG GTT TAG  TCT G  A2_ALK_Adapter_REV  GTG CTC TTC CGA TCT CCA GGC CTA GAA GAA  TGT TGG AGG T  A6_ALK_Adapter_FOR  GCG TTA TCG AGG TCG TTC CCC TAA TTG CCA  GAG AC  A6_ALK_Adapter_REV  GTG CTC TTC CGA TCT AAT AAA ACA GGG TTG  GTG GTG  A7_ALK_Adapter_FOR  GCG TTA TCG AGG TCC TTC TGT GTG CCT CTC  CAC A  A7_ALK_Adapter_REV  GTG CTC TTC CGA TCT ACC ATT TCA GCC ACC  TTG TC  B1_ALK_Adapter_FOR  GCG TTA TCG AGG TCT CCA TAA AGT TCT TGC  CCT CA  B1_ALK_Adapter_REV  GTG CTC TTC CGA TCT CAC CTC AGG AAT TCC  CAC TG  B2_ALK_Adapter_FOR  GCG TTA TCG AGG TCA TGG TAC ACA ATC TAA  TGG GTA TGC  B2_ALK_Adapter_REV  GTG CTC TTC CGA TCT GAG AGA GAG AAA GCT  CTG GTC TTT T  B3_ALK_Adapter_FOR  GCG TTA TCG AGG TCT TGA AGT CTT CCA GTG  TGC TG  B3_ALK_Adapter_REV  GTG CTC TTC CGA TCT TAA ACT CAT CAG GGC  AGC TTG  B4_ALK_Adapter_FOR  GCG TTA TCG AGG TCC TTG GTA GTT TAC CCT  CCT CCT C  B4_ALK_Adapter_REV  GTG CTC TTC CGA TCT AGG ATT AGG CAT TGA  TTG TGC TA  B5_ALK_Adapter_FOR  GCG TTA TCG AGG TCA TCT TGG CTG GTC CAT  CTG TAA  B5_ALK_Adapter_REV  GTG CTC TTC CGA TCT AGG ACC ACT GTC TAG  ACC AAG C  B6_ALK_Adapter_FOR  GCG TTA TCG AGG TCA GGT TTA AAA TGA GGG  AAA ATG G  B18_STAG2_Adapter_FOR  GCG TTA TCG AGG TCT AAA TCG GTC GGC TTC  CAT A  B18_STAG2_Adapter_REV  GTG CTC TTC CGA TCT GGT GAT GAG CTA TAG  TCA GGA GAA  B19_STAG2_Adapter_FOR  GCG TTA TCG AGG TCG AAG AAC ACC TTT TTG  CAA ATC T  B19_STAG2_Adapter_REV  GTG CTC TTC CGA TCT TTG ATG CCA GTT CCT  TTT GA  B20_STAG2_Adapter_FOR  GCG TTA TCG AGG TCT GAG CAT TGG TTT TGT  TTG C  B20_STAG2_Adapter_REV GTG CTC TTC CGA TCT AAA GAA CCA GAG CGC TGA ATT A CDS_APC_17nt_Adapter_FOR

Primer Name Primer Sequence
GCG TTA TCG AGG TCT TGA TGA AAG TTG ACT GGC GTA CDS_APC_17nt_Adapter_REV GTG CTC TTC CGA TCT CCT CAG GTT CTG GAA AAA TGT C  CDS_APC_23nt_Adapter_FOR  GCG TTA TCG AGG TCT GTT TGC TTG AGC TGC  TAG AAC  CDS_APC_23nt_Adapter_REV  GTG CTC TTC CGA TCT AGG CAA AAC AAA ATG  TGG GTA A  CDS_ATM_17nt_Adapter_FOR  GCG TTA TCG AGG TCG CCA ATA TTT AAC CAA  TTT TGA CC  CDS_ATM_17nt_Adapter_REV  GTG CTC TTC CGA TCT CCT GCT TGA CCT TCA  ATG CT  CDS_ATM_23nt_Adapter_FOR  GCG TTA TCG AGG TCG ATG AGG TGA AGT CCA  TTG CT  CDS_ATM_23nt_Adapter_REV  GTG CTC TTC CGA TCT GCC ATA CCT GTT TTC  CCA AT  CDS_KDM5C_18nt_Adapter_FOR  GCG TTA TCG AGG TCC CAC TTG GGA GGA TTC  TTC A  CDS_KDM5C_18nt_Adapter_REV  GTG CTC TTC CGA TCT CAG CCC TCC ATC ACC  TAC AT  CACNA1D_Adapter_FOR  GCG TTA TCG AGG TCG GTC CGA CTC AGG AGA  TGA A  CACNA1D_Adapter_REV  GTG CTC TTC CGA TCT TTT TTA GGG GAA GCC  CAA GT  PPP1R12C_Adapter_FOR  GCG TTA TCG AGG TCG GAC TCC TGC TTC ACA  TCG T  PPP1R12C_Adapter_REV  GTG CTC TTC CGA TCT CAC AAT GCA CCT GGG  TAA TG  C1_WDR5_Adapter_FOR  GCG TTA TCG AGG TCT CTC TGC AAA GTG GGT  GTT G  C1_WDR5_Adapter_REV  GTG CTC TTC CGA TCT CAT CGG AAT CCC AGA  AGC TA  C2_COPA_Adapter_FOR  GCG TTA TCG AGG TCT GTG ACT AGG CCA CAC  CAA C  C2_COPA_Adapter_REV  GTG CTC TTC CGA TCT CTG CAT GGG TGA ACA  AGA AC  C3_STAG2_Adapter_FOR  GCG TTA TCG AGG TCT TTC CCT GTA TAC CAA  TCA AGA CA  C3_STAG2_Adapter_REV  GTG CTC TTC CGA TCT TGG CAC GTA AAA GGT  GCT AAG TA  C4_STAG2_Adapter_FOR  GCG TTA TCG AGG TCT GGT GAT CCT AAC TGC  CCA TA  D9_HDAC2_Adapter_FOR  GCG TTA TCG AGG TCT CAT CAT GGT GAT GGT  GTT GA  D9_HDAC2_Adapter_REV  GTG CTC TTC CGA TCT TTC AAT TCT GCA TAC  AAG TTT CA  D10_HDAC2_Adapter_FOR  GCG TTA TCG AGG TCT GGC ATC CAG GTT CCA  TTA T  D10_HDAC2_Adapter_REV  GTG CTC TTC CGA TCT TAC CCC CAT TTG TCT  GCT TC  D11_D12_GLUL_Adapter_FOR  GCG TTA TCG AGG TCG GCC AAG CTT CTC GTT  TCT