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Targeted gene deletion using DNA-free RNA-guided Cas9 nuclease accelerates adaptation of CHO cells to suspension culture Namil Lee, JongOh Shin, Jinhyung Park, Gyun Min Lee, Su-hyung Cho, and Byung-Kwan Cho ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00249 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 9, 2016

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ACS Synthetic Biology

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Targeted gene deletion using DNA-free RNA-guided Cas9

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nuclease accelerates adaptation of CHO cells to

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suspension culture

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Namil Lee1,Ŧ, JongOh Shin1, Ŧ , Jinhyoung Park1, Gyun Min Lee1, Suhyung Cho1,*,

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and Byung-Kwan Cho1,2,*

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1

Department of Biological Sciences and KI for the BioCentury, KAIST, Daejeon 305-

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701, Republic of Korea 2

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Intelligent Synthetic Biology Center, Daejeon 305-701, Republic of Korea

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*Correspondence and requests for materials should be addressed to S.C.

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(email: [email protected]) and B.K.C. (email: [email protected])

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Ŧ

These authors contributed equally to this work.

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Abstract

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Chinese hamster ovary (CHO) cells are the preferred host for the production of a

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wide array of biopharmaceuticals. Thus, efficient and rational CHO cell line

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engineering methods have been in high demand to improve quality and productivity.

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Here, we provide a novel genome engineering platform for increasing desirable

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phenotypes of CHO cells based upon the integrative protocol of high-throughput

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RNA sequencing and DNA-free RNA-guided Cas9 (CRISPR associated protein9)

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nuclease based genome editing. For commercial production of therapeutic proteins,

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CHO cells have been adapted for suspension culture in serum-free media, which is

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highly beneficial in productivity and economical aspects. To engineer CHO cells for

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rapid adaptation to suspension culture, we exploited strand-specific RNA-seq to

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identify genes differentially expressed according to their adaptation trajectory in

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serum-free media. More than 180 million sequencing reads were generated and

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mapped to the currently available 109,152 scaffolds of the CHO-K1 genome. We

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identified significantly downregulated genes according to the adaptation trajectory

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and then verified their effects using the genome editing method. Growth-based

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screening and targeted amplicon sequencing revealed that the functional deletions of

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Igfbp4 and AqpI gene accelerate suspension adaptation of CHO-K1 cells. The

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availability of this strand-specific transcriptome sequencing and DNA-free RNA-

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guided Cas9 nuclease mediated genome editing facilitates the rational design of the

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CHO cell genome for efficient production of high quality therapeutic proteins.

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Keywords: Chinese hamster ovary (CHO) cells; High-throughput RNA-seq; DNA-

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free CRISPR/Cas9; Genome-editing, Suspension culture

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Chinese hamster ovary (CHO) cells are the main platform that is frequently used for

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the industrial production of therapeutic proteins. Thus, with the tremendous number

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of media optimization protocols, CHO cell lines have been extensively engineered to

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increase productivity and to enhance quality of the products (e.g., glycosylation).1, 2

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In recent years, based on rapid advances in DNA sequencing technologies, a wide

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array of genome-scale measurement techniques have been developed and applied

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to accumulate -omics data for CHO cell. Those include genome sequences,3, 4

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transcriptome,5, 6 and proteomic data7, 8, which enabled a systematic understanding

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of the genetic networks in CHO cell.9 The systems biology approach toward

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integration of omics data with modeling approaches may elucidate the relationship

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between genotype and phenotype of CHO cells.

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In particular, genetic information of CHO-K1 cell enables genome

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engineering using site specific nucleases such as zinc-finger nucleases (ZFNs),10-12

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transcription activator-like effector nucleases (TALENs),13 and clustered, regularly

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interspaced short palindromic repeats (CRISPR) mediated Cas9 nuclease.14, 15 Since

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the CHO cell shows genetic instability and clonal variation,16 highly efficient genome

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engineering methods are demanded in order to avoid the time-consuming process

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for the selection of the best clone for the production of each therapeutic proteins. To

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this end, site specific nucleases have been used to induce double-strand breaks

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(DSBs) at a targeted CHO genomic locus.14 DSBs are repaired via genome repair

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systems such as homologous recombination (HR) or non-homologous end joining

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(NHEJ). NHEJ is an imperfect repair system which connects the broken sites with

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indel generation, and leads frameshift mediated gene knockouts.17 In contrast to

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ZFNs and TALENs, CRISPR/Cas9 system is a RNA guided site specific engineering

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method which is rapid, cost-effective and scalable.18-20 Currently, this system has 3 ACS Paragon Plus Environment


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been applied to CHO cells with a particular focus on glycosylation related genes

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which directly effects the quality of therapeutic proteins .21 However, plasmid or

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lentivirus based delivery of CRISPR/Cas9 system is sometimes not suitable for

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industrial application of engineered CHO-K1 cell, because the delivered plasmid or

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lentivirus fragment randomly integrated into undesirable sites in the genome.22, 23 As

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an alternative protocol, the delivery of preassembled Cas9 protein and sgRNA

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ribonucleoprotein (RNP) complex could be a better method to minimize integration of

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the DNA fragment into the host cell genome.24 Because the RNP complex is DNA

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free and is rapidly degraded by endogenous proteases of the host cell after delivery

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into the cell, it reduces the unexpected alteration of the host genome.22 In addition to

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this, there is no need for the selection of optimized promoters and codon

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optimization for Cas9 nuclease expression in vivo.25

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Here, we rationally engineered CHO-K1 cell by applying high-throughput

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RNA-seq and DNA-free CRISPR/Cas9 mediated genome engineering. For large

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scale commercial production of therapeutic proteins, adaptation of CHO cells to

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suspension culture in serum-free media is essential step, which has a number of

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advantages for large scale production such as simple purification of the desired

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product and improvement of biological safety.26 However, lack of genetic information

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of CHO cells has limited the rational genome engineering of CHO cells, and thus

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research has been focused on the time-consuming optimization of specific serum-

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free media for individual CHO cell lines.27, 28 Therefore, we performed strand-specific

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RNA-seq analysis of CHO cells according to adaptation trajectory to identify specific

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genes that responded to the suspension adaptation. To determine the effect of those

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genes in the suspension adaptation, we applied DNA-free RNA-guided Cas9

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nuclease for the first time. Growth profiling based screening results demonstrated 4 ACS Paragon Plus Environment

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that the functional deletions of AqpI and Igfbp4 accelerated suspension adaptation

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compared to the parental cell line. We demonstrate an efficient and fast rational CHO

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cell engineering method, which is highly applicable to any phenotype of CHO cells.

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Results and Discussion

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RNA-seq analysis of CHO-K1 cell adaptation to suspension culture

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To profile the transcriptome dynamics of the CHO-K1 cell according to suspension

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culture adaptation, CHO-K1 cells were adapted for 10 passages in serum-free media

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(SFM4CHO). After 72 hr cultivation of the cells inoculated at a concentration of 0.5 ×

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106 cells/mL in 30 mL SFM4CHO, cells were harvested at the mid-exponential phase

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and passed into fresh media at a same concentration. When the viable cell

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concentration was over 1.0 × 106 cells/mL in consecutive passes (3~4 passages),

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the inoculation concentration was serially decreased by 0.3 × 106 cells/mL and 0.2 ×

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106 cells/mL.7 Exponentially growing cells at passages 0, 2, 4, 6, and fully adapted

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cells at passage 10 were selected for transcriptome analysis (Figure 1A).

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Strand-specific RNA sequencing was then performed on total RNA isolated

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from the cells harvested at 5 time points during suspension adaptation trajectory. A

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total of 180 million sequencing reads were obtained with 15 to 24 million reads

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generated from each individual library. After trimming adapter sequences and

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removing sequences shorter than 15 nt, more than 93% of the reads were

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successfully mapped to 21,395 scaffolds, which is 95% of the draft CHO-K1 genome

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sequence (Supplemental Table 1). Note that the current release of the CHO-K1 cell

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draft genome is composed of 109,152 scaffolds and 265,786 contigs, representing a

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total length of 2.45 Gb. Length of scaffolds range from 200 bp to 8,779,783 bp.3, 4

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Unique mapped read numbers for each gene was normalized using the DESeq 5 ACS Paragon Plus Environment


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package in R.29 For the coding transcriptome, more than 18,000 genes showed the

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normalized expression values of 0.39 or more, representing 56% of the annotated

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genes that were transcribed at each time points. Calculation of pairwise correlation

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coefficients (>0.95) demonstrated a high degree of reproducibility between biological

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replicates (Figure 1B). In addition, principal component analysis (PCA) showed

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distinct localization of each condition, which indicates high correlation between

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biological replicates (Supplemental Figure 1A).30 Among them, a total of 1,596

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genes showed significant changes in expression at two or more time points (P-value

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< 0.05). Based on the error sum of squares (SSE) analysis,31 their expression

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patterns were clustered into 15 different clusters using the K-mean clustering

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algorithm (Supplemental Figure 1B). Clusters of genes showing up- (507 genes)

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and down- (377 genes) -regulated tendency were grouped accordingly. Further, gene

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expression dynamics according to adaptation trajectory indicates that up and

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downregulated genes were clustered appropriately (Figure 1C). The differences in

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fold changes between the up and downregulated clusters was statistically significant

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(P = 1.03 e-142). Among the clustered genes, a total of 356 differentially expressed

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genes (DEG) were categorized into functional groups using the KEGG database

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(Figure 1D).32 Cell growth, replication, and nucleotide metabolism categories were

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enriched in upregulated clusters. In particular, DNA replication machinery such as

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DNA polymerase (pola2, pold2, and pole2), clamp (pcna), and helicase (dna2)

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coding genes were highly upregulated (fold change range 1.86 to 2.66). It is likely

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that the up regulation of these growth related genes contribute to the growth

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recovery of the CHO cell during suspension adaptation.33, 34 On the other hand, the

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cellular community category, which contains focal adhesion-related genes, was

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enriched in downregulated clusters (Figure 1D). For example, actin related genes 6 ACS Paragon Plus Environment

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and zyxin,35 which are critical components for actin polymerization was down-

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regulated (fold change 0.17). This indicates that reduction of focal adhesion

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increases motility and growth of CHO-K1 cells in suspension culture.36

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Screening genetic targets for accelerating adaptation to suspension culture Conventional analysis of RNA-seq often uses fold change values to describe

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change in expression of individual genes. However, in case of low expression genes,

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fold change based analysis generates unacceptably high false positives.37 In order to

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avoid choosing a false positive as an engineering target, the first (adherent) and last

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(suspension) time points in RNA-seq data was visualized by MA plots. This revealed

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that low abundance transcripts appear on the left x-axis whereas high abundance

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transcripts are located right x-axis (Figure 2A).38 From this analysis, we could

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distinguish between genes that have high fold change and expression values, from

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false positives. As a result, five genes showing the highest and lowest mean values

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of fold change were selected. Igfbp4 (NCBI Gene ID: 100772587), Sulf2

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(100772828), Fos (100689418), Aqp1 (100773231), and Nr4a1 (100764218) were

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selected as the five most significantly downregulated genes. On the other hand,

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LOC100769145, LOC100753467, Col4a1 (100757740), Nrg4 (100757283), and

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Nppa (100774350) were selected as the five most significantly upregulated genes

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(Supplemental Figure 2). Though these genes have not been studied well in CHO-

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K1 cells, many are related with cancer development and metastasis. For example,

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Igfbp4 encoding the insulin-like growth factor binding protein (IGFBP) domain and a

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thyroglobulin type-I domain, consistently inhibits several cancer cells, and Aqp1

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encoding an aquaporin, which functions as a molecular water channel protein, is a

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potential cancer biomarker that promotes metastasis and progression of cancer.39, 40 7 ACS Paragon Plus Environment


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Based on the fact that the adaptation of CHO-K1 cells to suspension culture shows

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similar phenotypes as cells during metastasis of cancer,41 these cancer related

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genes may have critical functions in suspension adaptation.

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Functional deletion of target genes by DNA-free RNA-guided Cas9 nuclease After unveiling an organism’s genome sequence, the functions of annotated genetic

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elements are often determined by the changes in cellular phenotypes under less or

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no function (loss-of-function mutation) of the gene product of interest.42 To this end,

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the RNAi mediated knockdown method has been extensively used to delete the

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function of a gene product of interest.43 However RNAi regulates gene expression

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via either RNA degradation or translation inhibition (i.e., post transcriptional

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regulation), which thus inactivates the function of a gene incompletely. Furthermore,

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the knockdown efficiency is often inconsistent with the unexpected off-target

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effects.44 In contrast, the genome editing method alters the genetic code, resulting in

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a complete “knockout” of the target gene. Recently, site specific nucleases, including

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zinc finger nuclease (ZFN)10-12, transcription activator-like effector nuclease

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(TALEN)13, 17, and RNA-guided Cas9 nuclease19, 20, 45 have been developed as

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genome editing tools. Site-specific nucleases induce double-strand breaks (DSBs) in

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the target locus, which are then repaired via a genome repair system such as

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homologous repair (HR) or nonhomologous end-joining (NHEJ) system. Since the

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NHEJ is an error-prone mechanism, it often results in small insertions or deletions

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that can disrupt, or knockout the gene.17 Among genome-editing methods, in contrast

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to the ZFN and TALEN methods, which use protein-DNA interactions to target

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specific loci, RNA-guided Cas9 nuclease employs RNA-DNA base-pairing rules

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which is easier to design, highly specific, and well suited for multiplexed genome 8 ACS Paragon Plus Environment

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editing.18, 45 Although this system has been applied to CHO cell engineering in

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handful reports,14, 15 these studies used plasmid based delivery of CRISPR/Cas9

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system to cells, which may cause unexpected random integration of DNA fragment

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into the CHO cell genome.22, 23 The unknown genomic alteration is not acceptable for

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industrial applications of modified CHO-K1 cells.46 Therefore, we applied a DNA-free

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CRISPR/Cas9 system to CHO-K1 cells for the first time. Due to the rapid

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degradation of the delivered complex of purified Cas9 protein and sgRNA in vivo, this

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system has less off-target effects than the plasmid or lentivirus based CRISPR/Cas9

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systems.22 In addition, this system does not require optimization of the Cas9 protein

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codon and promoter for CHO-K1 cells.25 We purified recombinant Cas9 protein from

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the E. coli BL21 (DE3) strain harboring the plasmid pET28a/Cas9-Cys.25 The purified

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Cas9 protein was then mixed with in vitro transcribed sgRNAs to prepare RNP

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complexes, which targets essential portions of the engineering targets selected from

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RNA-seq analysis (Figure 2B, Supplemental Figure 3).

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First, DNA editing efficiency of RNP complexes was validated by using in

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vitro cleavage assay, which revealed that the complex cleaves target sites efficiently

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only in the presence of the sgRNA (Figure 2C). For example, the preassembled

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Cas9 protein and AqpI-targeted sgRNA complex cleaved 526 bp of the target site

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into 331 bp and 195 bp fragments with 83.1% indel frequency in vitro. Next, to

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investigate whether an RNP complex cleaves a target sequence in vivo, the complex

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was transfected into CHO cells via electroporation. After 72 hr post-transfection, we

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observed that the RNP complex induced indel mutations in the targeted loci in the

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presence of sgRNA, whereas no detectable NHEJ-associated mutations was

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observed in the control experiment (Figure 2D). Indel frequencies were 40~50%,

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which is similar in efficiency to plasmid based CHO-K1 CRISPR/Cas9 mediated 9 ACS Paragon Plus Environment


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engineering.14 This efficiency is also comparable to RNP mediated target

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mutagenesis in human cells including fibroblast and pluripotent stem cells.22, 47

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Furthermore, to determine the gene having the most critical knockout effect

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on the adaptation to suspension culture, a knockout library was generated by

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treating sgRNA mixtures for the selected genes with Cas9 protein. The same

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amounts (40 µg) of each sgRNA were pooled and transfected into CHO cells with

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Cas9 protein. As expected, the RNP complex induced mutations that were detected

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at all target sites (Figure 2E). Despite indel frequencies that were lower than those

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in single sgRNA treated samples, the method was quite effective (20~50%). For

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quantitative analysis of the editing efficiency, we applied the Bioanalyzer 2200 for the

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T7E1 assay. The Bioanalyzer is more sensitive compared to gel electrophoresis to

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quantify DNA and showed positive correlation with gel electrophoresis result.48 For

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instance, the indel frequency in the Fos gene from single and multiple sgRNA treated

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samples were 35.2% and 33.7%, respectively (Figure 2F).48 In conclusion, the RNP

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mediated method is much efficient in single and in multiplex engineering, compared

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with plasmid or lentivirus based CRISPR/Cas9 editing methods.47 RNP mediated

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knockout population, which contains single and multiple knockouts, was used to

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screen the critical knockout among the selected engineering target sites.

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Effect of genome engineering on the adaptability of CHO-K1 cell line in

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suspension culture

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Adaptation of CHO-K1 cells to suspension culture in serum-free media is essential

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step for large scale production of biopharmaceuticals.49, 50 However, due to lack of

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genetic information about suspension adaptation, many studies have been focused

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on the optimization of serum-free media for individual CHO cell line.27, 28 Although 10 ACS Paragon Plus Environment

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proteomic analysis has been performed to understand intracellular response of CHO

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cells to adaptation in serum-free media followed by the observation of changes in

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phenotypes by plasmid-based overexpression of the selected genes, genetic

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mechanism of suspension adaptation has not been systematically elucidated using

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genome engineering. In order to investigate the effect of targeted genome

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engineering on proliferation rate during adaptation trajectory, CHO-K1 cell

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populations that contain NHEJ-associated mutations in the target site were

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maintained in the serum-free media. The seeding cell number was serially

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decreased from 5 × 105 cells/mL to 3 × 105 cells/mL and 1 × 105 cells/mL when the

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viable cell concentration was over 1 × 106 cells/mL. If viable cell concentration was

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not over 1 × 106 cells/mL after 72 hr of cultivation, seeding cell number was not

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decreased.7, 27 As a result, Aqp1 and Igfbp4 sgRNA treated samples showed ~54%

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reduced adaptation time (6 days) compared to controls (Figure 3A). Compared to

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the previous study which reduced adaptation time (25~33%) by overexpression of

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heat shock proteins, our result indicated extremely reduced adaptation time (~ 2

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fold).7 Effects of Fos, Sulf2, Nr4a1 and multiple sgRNA treated samples were not

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clearly observed during adaptation to suspension culture. Taken together, this is the

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first study involving the enhancement of a desired phenotype of CHO-K1 cells by a

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DNA-free genomic sequence modification method (Figure 3B, 3C). As mentioned

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before, adaptation of CHO-K1 cells to suspension culture shows similar phenotype to

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cells during metastasis of cancer.41 In addition, functions of the target genes are

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highly related with cancer development or metastasis.39, 51, 52 Thus, knockouts of

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these genes may influence suspension adaptation by a cancer development-like

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mechanism. One of the target genes, Aqp1 is upregulated in hypoxia or high-density

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conditions, which are typical conditions for actively growing cancer cells.52, 53 11 ACS Paragon Plus Environment


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Accordingly, down regulation of AqpI during adaptation may be caused by

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suspension culture conditions, which are typically of lower density compared to

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adherent cultures. Aqp1 knockout may alter cell shape by disturbing water transport,

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and support cellular response of CHO cells in suspension culture conditions.53 Igfbp4

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is expressed in a variety of tumor cells where it inhibits IGF-mediated cell

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proliferation by altering the interaction of IGFs with their cell surface receptors 40, 51, 54

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Therefore, knockout of Igfbp4, which causes the IGF-mediated growth promotion,

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may support growth recovery of CHO cells in suspension culture. Even though the

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knockout effect of Fos, Sulf2, and Nr4a1 were insignificant, they are attractive

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targets for engineering because they are also related with cancer development in

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various ways. For instance, the Fos family proteins have tumor suppressive impacts

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and reduce metastasis that might at least partly derive from changed adhesive

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properties.53 Down regulation of these genes might influence suspension adaptation

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of CHO cells through combinatorial effects with other genes by unknown

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mechanisms.

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Deep sequencing analysis of DNA-free RNA-guided Cas9 nuclease mediated

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cleavage at target genomic loci

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Due to the rapid degradation of the RNP complex after delivery into the cell, indels

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generated by DNA-free RNA-guided Cas9 nuclease could not be retained according

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to adaptation trajectory.22 Thus, the dynamics of indel frequency is directly related

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with the function of the targeted gene knockout. If knockout is effective and

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accelerates adaptation time, the indel containing population would increase

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according to the adaptation trajectory.55 In order to determine the changes in indel

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frequency induced by DNA-free RNA-guided Cas9 nuclease according to the 12 ACS Paragon Plus Environment

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adaptation trajectory, a deep sequencing analysis was performed using the genomic

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DNA obtained from first and last passage of multiple sgRNA treated cells. We

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amplified 355~398 bp DNA fragments each containing sgRNA target sites using

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sequencing adapter linked primer sets, followed by multiplex high-throughput

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sequencing. Approximately 63,000~88,000 sequencing reads per each sample were

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obtained, which were then aligned with the wild type sequences of the target sites.

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As a result, 1.2~27% indel frequencies were detected at all target sites at the

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adaption trajectory, whereas control cells transfected only with Cas9 protein showed

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indel frequencies of 0.1~0.2%. Portions of the Aqp1 and Igfbp4 knockouts increased

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2.5 and 5.7 fold, respectively. On the other hand, portions of the Fos, Sulf2 and

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Nr4a1 knockouts were maintained or reduced according to adaptation strategy,

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which is consistent with the effect of AqpI and Igfbp4 knockouts on the adaptation

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(Figure 4). Taken together, AqpI and Igfbp4 knockouts, which accelerate adaptation

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time were enriched in the population while the non-effective knockouts diminished

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during suspension adaptation in serum-free media. These results supports the basic

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theory of evolution, where advantageous genome modifications should be enriched

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in populations.55

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Conclusion

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Suspension adaptation is an essential step for large scale production of therapeutic

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proteins.27 In this study, transcriptome dynamics during suspension adaptation of

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CHO-K1 cells was elucidated using high-throughput RNA-seq. According to the

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adaptation trajectory, we demonstrated that growth related genes were upregulated

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and focal adhesion related genes were downregulated. Among the differentially

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expressed genes, Fos, Sulf2, Igfbp4, Nr4a1 and Aqp1 were the most significantly 13 ACS Paragon Plus Environment


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downregulated genes through the suspension adaptation trajectory. Next, we applied

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DNA-free RNA-guided Cas9 nuclease to manipulate targeted gene knockouts. Direct

3

delivery of purified Cas9 and sgRNA complex into CHO-K1 cells, generated

4

approximately 40~50% indel frequencies at desired sites. This method is more

5

convenient and effective than conventional vector based CRISPR/Cas9 systems.25

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In addition, it has less side effects, because the RNP complex is degraded rapidly

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after delivery into the CHO-K1 cell.22 Growth-based screening and deep sequencing

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analysis confirmed that the knockouts of Igfbp4 and Aqp1 efficiently accelerated

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adaptation time of CHO-K1 cells in suspension culture.

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Elucidation of the molecular mechanism behind the effect of disrupting target

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genes on cellular adaptation awaits further studies regarding the transcriptome

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dynamics after the disruption. In addition, overexpression of upregulated genes by

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targeted site specific integration would further accelerate the adaptation time of

14

CHO-K1 cells in suspension culture in combination with targeted gene knockouts.

15

Conventionally, most of CHO cell related studies have been focused on media

16

optimization to maximize the productivity.11, 29, 43 However, no universal optimized

17

media is available.7 Besides, CHO cell line engineering was performed using only

18

well-known mechanisms due to lack of omics based understanding.11, 14 Here, our

19

results provide a novel and rational genome engineering platform for increasing

20

desirable phenotypes of CHO cells. We found engineering targets from

21

transcriptome analysis and verified the effect of the selected genes on the desired

22

phenotype by targeted genome editing method. The availability of this rational design

23

of CHO cell genome promotes efficient production of high quality therapeutic

24

proteins.

25 14 ACS Paragon Plus Environment

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1

Materials and Methods

2

Cell culture and adaptation to serum-free growth environments. CHO-K1

3

adherent cells (#ATCC-CCL-61) were cultured with Dulbecco’s modified eagle

4

medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine

5

serum (FBS; Invitrogen, Carlsbad, CA) at 37 oC in a humidified incubator (Panasonic,

6

Wood Dale, IL) with 5% carbon dioxide. The cells were cultured in 25 cm2 cell culture

7

treated flasks with filter caps (Thermo Fisher Scientific Inc., Waltham, MA). Cells

8

were released from the 25 cm2 flasks using trypsin-EDTA (Thermo Fisher Scientific)

9

and expanded in 75 cm2 flasks (Thermo Fisher Scientific). Exponentially growing

10

cells were inoculated at a concentration of 5 × 105 cells/mL into 125 mL Corning

11

Erlenmeyer flasks (Sigma-Aldrich, St. Louis, MO) containing 30 mL of serum-free

12

media (SFM4CHO; Thermo Fisher Scientific) with 10% FBS. After 72 hr of cultivation,

13

cells were harvested, centrifuged at 1,200 rpm for 5 min, re-suspended in the 30 mL

14

SFM4CHO media with no FBS at 5 × 105 cells/mL. The seeding cell number was

15

serially decreased by 3 × 105 cells/mL to 1 × 105 cells/mL, when the viable cell

16

concentration at 72 hr cultivation reached 1 × 106 cells/mL. Suspension culture was

17

performed on an orbital shaker (INFORS-HT, Bottmingen, Swizerland) at 150 rpm in

18

a humidified, 5% CO2 incubator, at 37 oC.

19 20

Strand specific RNA sequencing. Total RNA was isolated from 5 × 106 cells using

21

1 mL of TRIzol (Invitrogen, Carlsbad, CA). DNase I treatment was applied to remove

22

genomic DNA from the isolated total RNA at 30 oC for 30 min. Total RNA was further

23

purified by chloroform extraction and ethanol precipitation. The total RNA was

24

quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc.) 15 ACS Paragon Plus Environment


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1

and the quality of the isolated total RNA was checked by visualization using the

2

ExperionTM Automated Electrophoresis system (Bio-Rad, Hercules, CA) and by

3

measuring the A260/A280 ratio (>1.8) using the NanoDrop 1000 spectrophotometer.

4

rRNA was specifically removed using the Ribo-Zero kit (Epicentre, Madison, WI) and

5

confirmed by the ExperionTM Automated Electrophoresis System. Strand-specific

6

RNA sequencing libraries were constructed using the TruSeq Stranded mRNA LT

7

Sample Prep Kit (Illumina Inc., San Diego, CA) according to manufacturer’s

8

instructions. Briefly, the rRNA-depleted RNA samples were chemically fragmented

9

and reverse transcribed into cDNA. The ends of the cDNA samples were repaired,

10

and Illumina adaptors were ligated. After the ligated products were PCR-amplified,

11

the libraries were confirmed using the TOPO cloning kit (Invitrogen). Concentration

12

of the libraries was measured with a Qubit® dsDNA BR Assay Kit (Thermo Fisher

13

Scientific Inc.), and the quality of the libraries was determined with the ExperionTM

14

Automated Electrophoresis System. The quantified samples were sequenced on a

15

MiSeq sequencer, using the MiSeq Reagent kit v2 (Illumina Inc., San Diego, CA) in

16

single-read mode with read lengths of 150 nucleotides.

17 18

In vitro DNA-free RNA-guided Cas9 nuclease assay. The pET28a/Cas9-Cys

19

vector containing recombinant Cas9 protein was purchased from Addgene (plasmid

20

53261) and expressed in the BL21 (DE3) strain. His-tagged Cas9 protein was

21

purified using Ni-NTA agarose beads (Qiagen, Crawley, UK), dialyzed against 50 mM

22

Tris-HCl (pH8.0), 200 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 20% glycerol.

23

Oligonucleotides for the sgRNA (Supplemental table 2) were annealed and cloned

24

into the px330 vector, which was purchased from Addgene (plasmid 42230). Linear

25

DNA templates for in vitro transcription were constructed by PCR from the sgRNA 16 ACS Paragon Plus Environment

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1

cassette cloned px330 vector with T7 promoter containing primers (Supplemental

2

table 2). The sgRNA was in vitro transcribed by using the MEGA shortscript T7 kit

3

(Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions.

4

Transcribed RNA was purified by denaturing polyacrylamide gel extraction and

5

ethanol precipitation. Purified Cas9 protein (500 µM) was mixed with PCR products

6

containing the target sequence and sgRNA (500 µM) in NEB buffer 3. After 1 hr of

7

incubation at 37 oC, and 2 min at 95 oC, the digested DNA was analyzed using

8

agarose gel electrophoresis. To prepare the RNP complex, purified Cas9 protein (30

9

µg) was mixed with in vitro transcribed sgRNA (40 µg) and incubated for 10 min at

10

room temperature. 2 × 105 cells were transfected with the RNP complex using a

11

NeonTM Transfection System (Invitrogen, Carlsbad, CA) according to the

12

manufacturer’s protocol.

13 14

T7E1 assay. Genomic DNA was extracted using a Wizard Genomic DNA Purification

15

Kit (Promega, Madison, WI) according to the manufacturer’s protocol. PCR products

16

containing the target sites were generated by nested PCR from genomic DNA

17

(Supplemental table 2). PCR amplicons were denatured and annealed to form

18

heteroduplexes using a thermocycler and digested with T7 endonuclease I (NEB,

19

Ipswich, MA) for 20 min at 37 oC. The digested DNA fragments were analyzed by 2%

20

agarose gel electrophoresis.

21 22

Amplicon sequencing. Amplicons (300~400 bp) containing CRISPR target sites

23

were generated from genomic DNA using the Phusion High-Fidelity DNA polymerase

24

(Thermo Fisher Scientific Inc.). PCR products were purified using a MinElute PCR

25

purification kit (Qiagen) and re-amplified with primers containing adapter sequences. 17 ACS Paragon Plus Environment


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Amplicon concentration was measured using a Qubit® dsDNA BR Assay Kit.

2

Amplicon samples were then pooled for multiplexing. Finally, multiplex sequencing

3

was carried out on a MiSeq v.2 instrument using a 250 cycle sequencing kit,

4

according to the manufacturer’s protocol for a 250-bp paired-end analysis. After

5

mapping the sequencing reads to the wild type sequence, insertion or deletion

6

located around the Cas9 cleavage sites were considered as a signature of NHEJ

7

mediated mutagenesis.

8 9

Data analyses. Short read sequences were mapped to the CHO-K1 genome

10

scaffold3 using CLC genomics work bench (CLC Bio) with the following parameters

11

(mismatch costs = 2, length fraction = 0.5, and similarity fraction = 0.8). For the

12

analysis of differential expression of genes between adherent and serum-free media

13

adapted CHO-K1 cells, sequence reads mapped to each annotated gene were

14

counted and normalized using the DESeq algorithm in the program R.29 Fold

15

changes were computed as the ratio of the normalized mapped reads. In-house

16

scripts used for data processing are available on request. The sequencing data have

17

been deposited in the Gene Expression Omnibus (GEO) database under accession

18

number GSE75094.

19 20

Supporting Information

21

Additional figures, RNA-seq read data, and details of sgRNA design, are available in

22

the supporting information.

23 24

Acknowledgements 18 ACS Paragon Plus Environment

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1

This work was supported by the Intelligent Synthetic Biology Center for Global

2

Frontier Project (2011-0031962 to B.-K.C.), the Basic Science Research Program

3

(NRF-2013R1A1A3010819 to S.C.) and the Bio & Medical Technology Development

4

Program (NRF-2013M3A9B6075931 to B.-K.C.) of the National Research

5

Foundation (NRF), funded by the Ministry of Science, ICT & Future Planning.

6 7

Author contributions

8

B.-K.C. and S.C. conceived of, and supervised the study. N.L., J.S., G.M.L., S.C.,

9

and B.-K.C. designed experiments. N.L., J.S., and J.P. performed experiments. N.L.,

10

J.S., S.C., and B.-K.C. analyzed data and wrote the manuscript.

11 12

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Figure legends

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Figure 1. A transcriptome analysis of CHO cells during adaptation to serum

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free suspension culture. (A) Cell growth profile of CHO cells during adaptation to

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serum free suspension culture. Asterisks indicate RNA-seq sampling time points. (B)

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Reproducibility of RNA-seq results. The RNA-seq results were highly reproducible

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(R2 > 0.95). Calculation of the distance between RNA-seq results is based on

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Euclidean metric. In the label, the first number followed by the sample name

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indicates at which time point the sample was harvested during suspension

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adaptation trajectory, and the second number demonstrates duplicate at same time

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point. (C) Distribution of up and downregulated gene clusters. Red lines inside the

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box indicate median value of each cluster. Black closed circle at the bottom and top

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of the boxes are first and third quartiles respectively. (D) Significantly enriched

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KEGG pathways of upregulated and downregulated clusters. Orange and navy box

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indicates upregulated and downregulated clusters respectively.

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Figure 2. Genome engineering of target genes using DNA-free CRISPR/Cas9

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system. (A) MA-plot of RNA-seq data. Navy blue open circles indicate 1596 genes

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that showed significant change in expression according to adaptation (p-value
0.95). Calculation of the distance between RNA-seq results is based on Euclidean metric. In the label, the first number followed by the sample name indicates at which time point the sample was harvested during suspension adaptation trajectory, and the second number demonstrates duplicate at same time point. (C) Distribution of up and downregulated gene clusters. Red lines inside the box indicate median value of each cluster. Black closed circle at the bottom and top of the boxes are first and third quartiles respectively. (D) Significantly enriched KEGG pathways of upregulated and downregulated clusters. Orange and navy box indicates upregulated and downregulated clusters respectively. 200x156mm (300 x 300 DPI)

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Figure 2. Genome engineering of target genes using DNA-free CRISPR/Cas9 system. (A) MA-plot of RNA-seq data. Navy blue open circles indicate 1596 genes that showed significant change in expression according to adaptation (p-value < 0.05). Grey and orange closed circles indicates the five most significantly upregulated and downregulated genes, respectively. (B) sgRNA design of the 5 knockout target genes. Each cleavage site is indicated by an orange arrowhead. Orange letter indicates the PAM sequence at the target locus. (C) In vitro functional analysis of RNP complex. The arrows indicate the expected positions of DNA bands cleaved by the RNP complex. Indel frequencies were calculated from the band intensities. (D) T7E1 assay to detect mutations of single and (E) multiple sgRNA treated cells at 72 h post transfection. The arrows indicate the expected positions of DNA bands cleaved by T7E1. (F) T7E1 assay confirmation using a Bioanalyzer 2200. Indel frequency of Fos gene single and multiple sgRNA treated sample was measured by using a Bioanalyzer. Size of the bands and indel frequency are indicated on the digital gel and the electropherogram. 237x183mm (300 x 300 DPI)



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Figure 3. Adaptation profiles. (A) Igfbp4 and Aqp1 knockout, which accelerated adaptation time. (B) Fos, Sulf2, and Nr4a1 knockout, which had no obvious effect on adaptation. (C) Adaptation profiles of multiple knockout libraries. Growth profiles of only Cas9 treated CHO-K1 cells are also shown as a control. The seeding cell number was serially decreased by 3X105 cells/mL, and 1X105 cells/mL when the viable cell concentration at 72 h of cultivation was over 1X106 cells/mL. The red horizontal line indicates 1.0X106 cells/mL. 183x109mm (300 x 300 DPI)


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Figure 4. Targeted deep sequencing analysis of the 5 knockout target loci in multiple sgRNA treated CHO-K1 cells. White boxes and purple boxes indicates indel percent at initial, and final (passage 5) passage, respectively. Values above to arrows indicate fold changes. 86x70mm (300 x 300 DPI)


Targeted Gene Deletion Using DNA-Free RNA ... - ACS Publications

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