Generation of a DIC pig line
To flexibly induce the expression of SpCas9 in pigs, a binary third-generation tetracycline-inducible (Tet-On 3G) system, consisting of the rtTA and TRE3G-controlled SpCas9-T2A-tdTomato expressing cassette, was used (Fig. 1a). The PFFs carrying rtTA-expressing cassette in the porcine Rosa26 (pRosa26) locus (pRosa26-rtTA PFFs), but not including TRE3G-tdTomato-expressing cassette, were isolated from crossbreeding our previously generated Dox-inducible tdTomato-expressing pigs [31] (Additional file 1: Fig. S1a). A targeting vector containing TRE3G-driving SpCas9-T2A-tdTomato-expressing cassette and puromycin resistance cassette, flanked by 911 bp 5′ arm and 1088 bp 3′ arm from the porcine Hipp11 (pHipp11) locus, another confirmed safe harbor site [30], were designed (Additional file 1: Fig. S1b). With this vector, the utility of the 2A peptide could assure the uniform expression of SpCas9 and tdTomato, the single-cell-derived positive colonies could be selected by puromycin treatment, and the expression of SpCas9 could be displayed by the indicator of tdTomato. TRE3G-controlled SpCas9 vectors were inserted into the pHipp11 locus in pRosa26-rtTA PFFs by CRISPR/Cpf1-mediated HDR (Additional file 1: Fig. S1b). A total of 44 single-cell-derived colonies were picked after about 10-day puromycin selection. For these colonies, 38 were successfully expanded in 24-well plates and further screened by 5′- and 3′- junction fragment PCR. The PCR results showed that 22 colonies carried TRE3G-SpCas9-T2A-tdTomato elements in the pHipp11 locus (Additional file 1: Fig. S1c, d). Two representative cell colonies (1# and 23#) and re-constructed SCNT embryos generated by using these cells as donors were treated with Dox for 3 days. The tdTomato fluorescence was observed in Dox-treated cell colonies and re-constructed embryos under an inverted fluorescence microscope, whereas no tdTomato fluorescence was found without Dox induction (Additional file 1: Fig. S1e, f). When reconstructed embryos were cultured for 6 days, almost no difference of blastocyst rates between Dox-treated (19.5%, 40/205) and Dox-untreated (20.4%, 23/113) groups was observed, while the previous report showed that zygote injection of RNA encoding Cre recombinase resulted in reduction of blastocyst rate [26], indicating that Dox-dependent SpCas9 expression may have less negative impact on porcine embryo development than Cre recombinase expression. These results suggested that the established DIC system in pigs could result in Dox-dependent SpCas9 expression at the cellular and embryonic level.
Next, five positive cell colonies (1#, 14#, 23#, 24#, and 29#) were pooled together and used as the nuclear donors for SCNT. A total of 809 reconstructed embryos were generated and then surgically transferred into four surrogate mothers (9602, 0101, 1106, and 2804). Two surrogates (50%, 2/4, 9602 and 0101) were confirmed to be pregnant and developed to full term. After about 113 days of gestation, six male cloned piglets (9602-1#, 9602-3#, 9602-5#, 0101-1#, 0101-3#, and 0101-5#) were delivered naturally (Fig. 1b, c). The 5′- and 3′-junction fragment PCR showed that two piglets (9602-3# and 0101-3#) carried heterozygous TRE3G-SpCas9-T2A-tdTomato-expressing cassette and one piglet (0101-1#) harbored homozygous TRE3G-SpCas9-T2A-tdTomato-expressing cassette in the pHipp11 locus (Fig. 1d; Additional file 1: Fig. S1a, b). All six cloned pigs carried heterozygous rtTA-expressing cassette in the pRosa26 locus. Except for 0101-3#, which was born weak and died early, the other two piglets (9602-3# and 0101-1#) were healthy and grew up to adult age without overt abnormalities (Fig. 1c). The low cloning efficiency and the perinatal death might be due to overall inefficient SCNT procedures as that previously reported [32], rather than genetic modification of donor cells per se. The porcine ear fibroblasts (PEFs) were isolated from the ear tissues of the cloned piglets and cultured in the medium with or without Dox. The tdTomato fluorescence was observed under an inverted fluorescence microscope (Fig. 1f), and approximately 51.2% DIC-PEFs were tdTomato positive by fluorescence-activated cell sorting (FACS) analysis (Fig. 1e). No tdTomato fluorescence was observed in DIC-PEFs without Dox treatment and pRosa26-rtTA PEFs with and without Dox treatment. SpCas9 proteins were further assayed by immunofluorescence staining and Western blot. The results showed that SpCas9 and tdTomato proteins were uniformly expressed in DIC-PEFs with Dox induction (Fig. 1f, g). These results suggested that the expression of SpCas9 and tdTomato was consistent and depended on Dox presence in pigs in vitro.
By crossing the two male DIC founders (9602-3# and 0101-1#) with six wild-type sows, 39 of F1 pigs were delivered. Of these offspring, 11 simultaneously carried rtTA-expressing cassette and TRE3G-SpCas9-T2A-tdTomato-expressing cassette in the pRosa26 and pHipp11 loci, respectively (Additional file 2: Table S1). The established DIC transgenic pig line was used for further study.
Establishment of Dox administration protocol for inducing SpCas9 expression and assessment of SpCas9 expression level in different organs of DIC pigs
To establish Dox administration protocol for inducing SpCas9 expression in vivo, we first treated the DIC pigs with Dox via oral administration. When DIC piglets were orally administrated with low dosage of Dox (25 mg/kg/day), weak tdTomato fluorescence was observed in most organs through using goggles in a dark room with appropriate excitation and emission filters. After oral administration of high dosage of Dox (50 mg/kg/day), tdTomato fluorescence was obviously increased (Additional file 1: Fig. S2). These results indicated that the expression level of SpCas9 in vivo depended on Dox dosage. However, only about 20% of peripheral blood mononuclear cells (PBMCs) (18.1% for monocytes, 43.8% for granulocytes, and 57.5% for lymphocytes) were tdTomato positive after one-week oral administration with high dosage (Fig. 2a). Next, we further verified whether oral administration combined with intraperitoneal injection could improve the induction efficiency in vivo. The FACS results of peripheral blood showed that more than 80% PBMCs (78.2% for monocytes, 83.7% for granulocytes, and 88.3% for lymphocytes) expressed the tdTomato fluorescence, suggesting that oral administration combined with intraperitoneal injection of Dox could significantly increase the induction efficiency (Fig. 2a). Immunohistochemistry (IHC) staining results also confirmed that a wide range of induction of SpCas9 expression in many tissues and organs, when compared with oral administration of Dox only (Additional file 1: Fig. S3). Therefore, oral administration combined with intraperitoneal injection was selected as the standard method to deliver Dox in the following experiments.
Next, we detected the expression levels of SpCas9 in different organs after treatment with Dox by quantitative PCR (Q-PCR), Western blotting, and immunofluorescence staining. These results showed that the SpCas9 protein were expressed in all assessed organs but displayed different levels in different organs (Fig. 2b, c, d). Among the tested organs, the pancreas, stomach, intestine, lung, and spleen showed high expression levels of SpCas9, whereas the brain, skeletal muscle, liver, and heart showed low expression levels. The relative low expression of SpCas9 in the brain and liver was possibly due to Dox inaccessibility and degradation, respectively. For the heart and skeletal muscle, possibly the low rtTA expression due to the weak activity of endogenous Rosa26 promoter in these organs, the same as previously reported in mice [33], might account for the low SpCas9 expression. These results indicated that broad and tightly controlled SpCas9 expression was achieved in DIC pigs in vivo through Dox treatment.
Time-controlled SpCas9 expression at the embryonic stage is very important for lineage tracing and embryonic conditional gene knockout. Therefore, we next verified whether the expression of SpCas9 could be efficiently induced at the embryonic development stage by treating pregnant sows with Dox. The DIC founder, 0101-1#, was cross-mated with one wild-type sow, and the pregnancy status was confirmed by B-ultrasound. The sow at 42 days of gestation was fed with Dox for 6 days combined with three intravenous injections every other day (Fig. 2e). The sow at 48 days of gestation was sacrificed to retrieve DIC fetuses. Twelve fetuses were obtained, and six were observed with high level of tdTomato fluorescence in the whole body by using goggles with appropriate excitation and emission filters (Fig. 2f). The brain, heart, stomach, lung, intestine, liver, and gonad were collected for further analysis. Obvious tdTomato fluorescence was observed in all collected organs (Fig. 2h). Q-PCR and IHC staining results also suggested broad SpCas9 expression in DIC fetuses with Dox induction (Fig. 2g, i). Notably, the fetal brain and liver expressed high levels of SpCas9 and tdTomato, which were different from adult DIC pigs in vivo, possibly due to the absence of the blood-brain barrier and less developed liver function in 48-day-old porcine fetuses. These results confirmed that time-dependent tight in vivo control of SpCas9 expression can be broadly achieved in most organs of fetuses through treatment of pregnant DIC sows with Dox.
Validation of the DNA cleavage activity of Dox-induced SpCas9 protein for genomic editing ex vivo
To validate the DNA cleavage activity of Dox-induced SpCas9 protein ex vivo, DIC-PFFs were isolated from 35-day-old fetuses retrieved from a pregnant wild-type sow mated with the 0101-1# DIC founder. Six fetuses were obtained, and two fetuses (DIC-F1-3 and DIC-F1-4) simultaneously carried heterozygous knock-in alleles at the pRosa26 and pHipp11 loci, respectively (Additional file 1: Fig. S4a). The remaining four fetuses (DIC-F1-1, DIC-F1-2, DIC-F1-5, and DIC-F1-6) only carried heterozygous TRE3G-SpCas9-T2A-tdTomato-expressing allele at the pHipp11 locus. The Western blotting and immunofluorescence staining results showed that the expression of SpCas9 was tightly controlled by Dox in DIC-F1-PFFs (Additional file 1: Fig. S4b-d). Through continuous stimulation with Dox, the percentage of tdTomato-positive PFFs gradually increased from day 1 to day 6. Following withdrawal of Dox, the percentage of tdTomato-positive PFFs gradually decreased (Additional file 1: Fig. S4e, f).
A total of 8 sgRNA-expressing vectors targeting porcine TP53, APC, KRAS, OCT4, LMNA, ALK, EML4, and PCSK9 genes, respectively (Fig. 3a), were designed and transfected into DIC-F1-PFFs, which were then cultured in the medium supplemented with Dox. Sanger sequencing results of the PCR products corresponding to amplified targeted sites showed that induced SpCas9 could cut the porcine genome for all tested targeted sites (Additional file 1: Fig. S5). Quantitative assessment of Sanger sequencing results showed that the efficiency of genome editing ranged from 4.5% to 64.3% (Fig. 3b). Next, we further investigated whether the DIC system could be used to achieve large-scale chromosome genetic engineering, such as chromosome inversion and large fragment deletion (Fig. 3c). The tested ALK-sgRNA and/or EML4-sgRNA were transfected into DIC-F1-PFFs. PCR and Sanger sequencing results showed that the events of chromosome inversion and large fragment deletion were successfully induced in Dox-treated DIC cells with simultaneous delivery of ALK-sgRNA and EML4-sgRNA (Fig. 3d). RT-PCR and subsequent Sanger sequencing results of TA clones showed that the EML4-ALK fusion transcripts were also expressed in these cells (Fig. 3e). In addition, the cascade architecture of ALK-sgRNA linked EML4-sgRNA by pre-tRNA sequence could also result in chromosomal inversion and large fragment deletion in DIC-F1-PFFs with Dox treatment with comparable efficiency (Fig. 3d). These results confirmed that Dox-induced SpCas9 can engineer the porcine genome and generate the indels, chromosomal inversions, and large fragment deletions with specific sgRNAs.
Targeted transcriptional activation of porcine endogenous genes by using the DIC system
Targeted gene activation is of critical importance for developing therapeutic targets and fundamental biology studies. However, endogenous target gene activation is difficult to implement. The CRISPR/Cas9-based system has been repurposed to enable enhanced gene expression or gene activation, which always relies on sgRNA to recruit dCas9 linked by transcriptional activation complexes to target loci [34]. To expand the applicability of the DIC system beyond genome editing, we next tested whether endogenous gene activation could be implemented through combination of dead sgRNA containing MS2-loop and transcriptional activation complexes [35, 36]. A cassette containing transcriptional activation complexes MS2-P65-HSF1 (MPH) and series engineered sgRNAs with additional MS2-loop was designed (Fig. 3f). To avoid the double stranded breaks created by the SpCas9 and sgRNA complex, modified sgRNAs carrying short target sequence (14-bp) were chosen to inactivate SpCas9 [35]. When Dox-treated DIC cells are transfected with plasmids expressing MPH activators and modified sgRNAs, the transcriptional machinery can be recruited to target loci by SpCas9 protein and MS2-looped sgRNA complex, and then activate or enhance the mRNA transcription, thereby increasing the expression levels of target genes (Fig. 3g). As proof-of-principle experiments, we designed and constructed six modified sgRNAs (dgRNA-1, dgRNA-2, dgRNA-3, dgRNA-4, dgRNA-5, and dgRNA-6) located within 100 bp upstream of the translational start site for the CDX2 and SOX2 loci, respectively (Fig. 3h, i). Six modified sgRNAs in tandem were also created to elicit stronger gene activation through previously reported golden gate assembly [37] (Fig. 3f). For the CDX2 locus, the Q-PCR results suggested that all six pCDX2-dgRNAs can be used to increase the transcript level with 1.74- to 170.10-fold change. Up to 187-fold change was achieved using the six modified sgRNAs in tandem (Fig. 3h). For the SOX2 locus, only pSOX2-dgRNA-4 and dgRNA-6 alone showed the ability to increase the gene expression with 3.8-fold change and 2.8-fold change, respectively. However, when the six dgRNAs were used in tandem, 15.1-fold change of SOX2 expression was achieved (Fig. 3i).
Development of a facile spatiotemporal gene knockout strategy based on the DIC system
The DIC pig model allows flexible temporal control of SpCas9 activity in pigs via simple chemical induction at a specific time in vivo and in vitro but does not enable spatial-control of SpCas9 activity directly. Interestingly, on the basis of the DIC pig model, a construct containing 2A-linked rtTA-pA and one or multiple U6-sgRNAs cassette could be inserted into the downstream of tissue-specific expressing genes of PFFs carrying Hipp11-TRE3G-Cas9-T2A-tdTomato allele, which allows one-step generation of germline-inherited pigs enabling not only temporal but also spatial control of gene function in vivo under chemical induction (Fig. 4a, b). As a proof-of-principle experiment, the pancreas was selected as the first target organ to perform spatiotemporal gene knockout given that the uniformly high expression level of SpCas9 was observed in the pancreas derived from DIC pigs treated with Dox. Previous studies have found that GATA4 and GATA6 transcription factors in the pancreas play a key role in neonatal diabetes [38]. However, the function of GATA4 and GATA6 transcription factors in the pancreas remains elusive and appears different dosage sensitivity between humans and mice [38,39,40]. Therefore, we constructed three transgene cassettes to achieve spatiotemporal knockout of GATA4 or GATA6 or simultaneous knockout of GATA4 and GATA6 (Fig. 4c). We screened and selected sgRNAs for targeting the PDX1, GATA4, and GATA6 loci in PFFs (Additional file 1: Fig. S6a-c). By using fibroblasts carrying TRE3G-driving SpCas9-T2A-tdTomato expression cassette at the pHipp11 locus, we picked 198, 268, and 257 single-cell derived colonies, respectively. For these colonies, 90, 78, and 143 were successfully expanded in 24-well plates and further screened by 5′- and 3′- junction fragment PCR. Genotyping PCR results showed that 46 (46/90, 51.1%), 51 (51/78, 65.4%) and 7 (7/143, 4.9%) colonies carrying transgene integration were successfully generated for GATA4, GATA6, and GATA4 and GATA6, respectively (Fig. 4d; Additional file 1: Fig. S6d-f). When these cells were used as donor nuclei for SCNT, spatiotemporal knockout of GATA4 or GATA6 or simultaneous knockout of GATA4 and GATA6 pig lines can definitely be obtained through only one-step SCNT cloning in the future.
Validation of the effectiveness of in vivo genome editing and generation of metastatic PDAC model by using DIC pigs
We next verified whether in vivo genome editing could be carried out by delivering sgRNA into DIC pigs accompanied with Dox induction. Given that the expression levels in the pancreas were relatively high after Dox induction, the pancreas was selected as the objective organ to model PDAC through mutating the tumor suppressor genes in vivo. AAV vectors containing sgRNAs targeting two tumor suppressor genes, pTP53 and pLKB, as well as human KRASG12D expressing cassette, referred to as AAV-PKL, were constructed (Fig. 5a). Compared with AAV8 and AAV9, AAV6 could efficiently and safely target both normal and neoplastic pancreases via retrograde ductal delivery without inducing pancreatitis [41]. The AAV-PKL vectors were used for packaging AAV6-PKL. Four DIC-F1 piglets (DIC-F1-82, DIC-F1-86, DIC-F1-87, and DIC-F1-92; one for 55-day-old and three for 43-day-old) were injected with 2 × 1012 genome copies (GC) of AAV6-PKL into the main pancreatic duct and/or the body of the pancreas (Fig. 5b). One DIC-F1 piglet (DIC-F1-88) injected with equivalent volume of 0.9% saline was used as the control. The detailed information for injection and animals is summarized in Additional file 2: Table S1. Dox administration was performed at day 16 to day 24 post AAV-injection via oral administration combined with three intravenous injections every other day. To verify whether the predicted pLKB1 and pTP53 gene editing could be simultaneously achieved by AAV6-PKL delivered sgRNAs combined with Dox induction, these five piglets were sacrificed to harvest the whole pancreases after 14 weeks of AAV6 or saline injection. Each pancreas was then randomly dissected to 15 pieces (named as P1-P15 indicated in Additional file 1: Fig. S7a) and subjected to amplicon deep sequencing. The sequencing results showed that 11 (18.3%, 11/60) samples harbored gene editing at the pTP53 and/or pLKB1 loci, whereas gene editing was almost undetectable in DIC-F1 pigs injected with 0.9% saline. Of these 11 samples, 10 had indel mutations at the target site of pTP53-sgRNA with efficiencies ranging from 3.62% to 11.57%; 5 harbored indel mutations at the target site of pLKB1-sgRNA with efficiencies ranging from 7.41% to 15.56%; 4 simultaneously incorporated indel mutation at the pTP53 and pLKB1 loci (Fig. 5c; Additional file 1: Fig. S7a). We further analyzed the type and ratio of indel reads. The representative top 5 indel reads were listed in Fig. 5d, e and Additional file 1: Fig. S7b-j. We found that pLKB1-sgRNA and pTP53-sgRNA could induce more frequent out-of-frame mutation than in-fame mutation. More specifically, sgRNA targeting the pLKB1 locus induced a large proportion of mutations with 3N + 1 or 3N + 2 bp insertions or deletions, whereas sgRNA targeting the pTP53 locus induced predominant mutations with 3N + 1 bp insertions or deletions. These results suggested that Dox-induced SpCas9 can efficiently introduce target gene mutation with specific sgRNAs in vivo.
We further injected AAV6-PKL to three other DIC-F1 pigs with different dosages (3.5 × 1012 GC for DIC-F1-96, 3.0 × 1012 GC for DIC-F1-99, and 1.5 × 1012 GC for DICK-F1-1) (Additional file 2: Table S1) and raised the pigs for long term to determine whether pancreatic cancer could develop. Dox administration was performed at day 9 to day 17 post AAV injection via oral administration combined with three intravenous injections every other day (Fig. 5a). DIC-F1-99 pig died during Dox administration due to unknown reasons (Additional file 2: Table S1). The health status of the remaining two pigs was closely monitored by visual inspection and weight tracking. For DIC-F1-96, obvious abdominal distension and decreased ingestion were observed at about 21 weeks post AAV6 injection. Positron emission tomography-computed tomography (PET-CT) was performed for DIC-F1-96. The PET-CT results revealed that a huge mass in the abdominopelvic cavity and massive ascites; pancreatic metabolism was diffusely slightly high (Fig. 5f). DIC-F1-96 was then euthanized (Additional file 1: Fig. S8a). During autopsy, hyperplastic and adherent neoplasms in the liver, large intestine, diaphragm, and small intestine and enlarged lymph nodes were also found (Fig. 5g). These results suggested that pancreatic malignant tumor with multiple lymph node metastasis in the abdominal cavity possibly developed in DIC-F1 pig through in vivo genome editing.
Given that we initiated porcine tumor formation through local injection of AAV6-PKL restricted to the main pancreatic duct to drive the transformation of ductal epithelial cells, we first evaluated whether PDAC was developed at the local injection site. Hematoxylin-eosin (H&E) staining results of the pancreatic head near the local injection site indicated that the pancreatic histological structure was disorganized, and nodular tumors occurred around the pancreatic duct (Fig. 6a). Upon histological staining of geographically separate regions, we found that porcine PDAC samples displayed typical lumenal masses with dominantly cellular atypia, which was consistent with the morphological features of human patients with PDAC. Porcine tumor sections were also extensively stained positive with Masson’s trichrome (Additional file 1: Fig. S8c), indicating that a desmoplastic tumor stroma developed similar to human patients with PDAC [42]. IHC staining of pancreatic head and tumor sections showed that pERK (a KRAS activation effector), PCNA (a proliferative marker), CK19 (a ductal origin marker), E-cadherin (an epithelial lineage marker), and vimentin (a mesenchymal marker) were expressed in the tumor stroma (Fig. 6a, b), indicating that PDAC had developed in the DIC pig. Dual immunofluorescence staining on PDAC samples showed that CK19-positive regions were also positive for proliferating marker, PCNA (Fig. 6c). Considering that pancreatic stellate cells are the critical mediator of tumor-associated fibrosis, we also assayed the expression of pancreatic stellate cell marker αSMA. The expression of αSMA was associated with areas of CK19-positive PDAC and exclusively localized to the tumor stroma (Fig. 6c). Similarly, pancreatic tumors from other separate areas also expressed E-cadherin, pERK, and vimentin in the tumor stroma (Additional file 1: Fig. S8b). Metastatic tumors in the liver, intestine, diaphragm, and enlarged lymph nodes were also sectioned and subjected to histological analysis. H&E staining results indicated that tumor cells were occasionally accompanied with invasion of inflammatory cells in the diaphragm, lymph node, liver and intestine (Fig. 6d). Severe tissue fibrosis occurred in neoplasms and adherent organs confirmed by Masson’s trichrome (Additional file 1: Fig. S8c). Consistent with primary pancreatic tumors, IHC staining was positive for PCNA and pERK in these metastases. CK19 expression could also be detected in metastatic tumor cells, but not in hepatocytes, lymphocytes or muscle cells (Fig 6e–h). Human KRASG12D expression was detected in all six analyzed pancreatic tumors through Q-PCR experiments with mutant KRAS specific primer pairs (Fig. 7a), but not in DIC control pig without AAV6-PKL injection.
To further evaluate the induced gene mutation at targeting sites, genomic DNAs were extracted from pancreatic head containing AAV6 injected sites (Pancreas-I), pancreatic tumors (Pancreas-T), metastatic tumors in liver (Liver-T), small intestine (Small intestine-T), large intestine (large intestine-T), tissues adjacent to tumors (Pancreas, Liver, Small intestine, Large intestine), and tissues from the diaphragm, thymus, and lymph node. We collected 3 to 6 samples of each group for statistical analysis. Next-generation sequencing (NGS) of PCR-amplified pLKB1-sgRNA and pTP53-sgRNA targeting sites using these genomic DNAs as template was then conducted. Indels at pLKB1 and pTP53 targeting sites were found in all tested tissues except that from un-induced DIC control pig (Fig. 7b). Indel frequency in tumors was obviously higher than that in the pancreas and liver adjacent to tumors. The highest indel frequency reached 37.1% for pLKB1 and 35.8% for pTP53 in Pancreas-I. Interestingly, comparable indel frequency in tumors from the small/large intestine and in adjacent tissues was found, indicating a high ratio invasion of metastatic tumor cells into intestines. The thymus and lymph node with detectable indel could be another direct evidence of malignant tumors accompanied with lymph node metastasis (Fig. 7b). By statistically analyzing indel reads, small deletion (1–3 bp), large deletion (> 50 bp), and small insertion (1–3 bp) were the dominant mutation types for the LKB1-sgRNA targeting site (Fig. 7c), whereas large deletion (> 50 bp) and small insertion (1–3 bp) were the dominant mutation types for the pTP53 targeting site (Fig. 7d). Although the indel sizes and positions varied between each group, we detected the most variable mutation types in pancreatic tissues, and the most frequent mutant DNA base pair covered the predicted SpCas9 cleavage sites, both for the pLKB1 and pTP53 loci (Fig. 7e, f). Combining statistical heatmap and mutation type analysis, several indel reads incorporating specific mutation types, such as insertion/deletion 1 bp mutation at pLKB1 targeting site and deletion 92 bp at pTP53 targeting site, were enriched during tumor formation and malignancy (Fig. 7e–g). Of particular note is the specific mutation type of deletion 92 bp at pTP53 targeting site, which had predominant enrichment among varied indel reads in most of detected tumor and adjacent tissues (Fig. 7e–g). These enriched indel reads possibly reflect specific mutation types which can empower tumor cells with survival, proliferative, and metastatic advantages.