Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells

Yilan Zhang 1&, Xianglian Ge1&, Fayu Yang1, Liping Zhang1, Jiayong Zheng2, Xuefang Tan3, Zi-Bing Jin1, Jia Qu1, and Feng Gu1*

1School of Ophthalmology and Optometry, EyeHospital, WenzhouMedicalUniversity, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027 China

2Department of Gynecology and Obstetrics, People’s Hospital of Wenzhou, Wenzhou, Zhejiang 325000 China

3Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH),Chinese Academy of Sciences,Guangzhou, Guangdong510530 China

*Correspondence to: Feng Gu, Center for Vision Research, Eye Hospital, Wenzhou Medical University, China; 270 Xueyuan West Road, Wenzhou, Zhejiang, China, 325027 Phone (Fax): +86-577-8806 7926; E-mail:

These two authors contributed equally to this paper

Table of Contents

Supplementary methods

Supplementary Figures

Figure S1

Figure S2

Supplementary Tables

Table S1

Table S2

Table S3

Supplementary methods

The detection of GFP gene copy number

GFP gene copy number was measured by Q-PCR. Briefly, the genomic DNA was isolated from the cells with GFP gene (SC1) or without GFP (Parental 293 cells) by standard techniques. Three pairs of PCR primers for the amplification of the fragments of GFP, ACTB (Beta-ACTIN) and FRMD7 genes were designed with online program ( respectively. ACTB (NM_001101) and FRMD7 (NM_194277) were located at autosomal chromosome and X chromosome, respectively, there are two ACTB andthreeFRMD7 gene copies per cell( Sequences of primers used in this study are shown in Table S3. For quantitative PCR, the PCRs were performed as 95°C for 10 minute, followed by 40 cycles of 95°C for 10 second and 60°C for 60 second. Q-PCR data was analyzed with StepOne software. The GFP relative copy number were normalized toACTB or FRMD7 using the formula 2^-(CT GFP- CT ACTB)/2^-(CT FRMD7- CT ACTB) or 2^-(CT GFP- CT FRMD7)/2^-(CT ACTB-CT FRMD7). Data shown are obtained from biological triplicates.

Figure S1. GFP gene copy number measured by Q-PCR

The GFP relative copy number was normalized to ACTB or FRMD7, and the gene copy number ratios of GFP to ACTB and to FRMD7 are about 0.42, 0.34, respectively. The results indicated that there is one GFP gene per cell.

Figure S2. Sequencing of GFP-negative colony from NGA PAM of CRISPR/Cas9 -mediated DNA cleavage

The GFP negative colonies were picked individually from NGA PAM of CRISPR/Cas9 -mediated DNA cleavage and the whole GFP gene was sequencing. Compare to wild-type (WT), one colony (mutant) has an 11-bp deletion, including one nucleotide of PAM (A of TGA) and 10-nucleotide following sequence (A, B), which leads to frame-shift mutation of GFP gene. It further confirmed that mutation does generate from NGA PAM for CRISPR/Cas9 -mediated DNA cleavage at GFP locus. Target sequence is in blue and PAM in pink.

Table S1. Target sequences.

Target sites for CRISPR/Cas9 System with NGG PAMs.

Target site ID / Target sequence
(5'-3') / PAM / Strand
NGG-0 / CAAGTTCAGCGTGTCCGGCG / TGG / -
NGG-1 / CAAGTTCAGCGTGTCCGGCG / AGG / +
NGG-2 / CGCGCCGAGGTGAAGTTCGA / GGG / +
NGG-3 / CAAGATCCGCCACAACATCG / AGG / +

Table S2. Target sequences .

Target sites for testing PAM specificity in CRISPR/Cas9 system.

Target site ID / Target sequence
(5'-3') / PAM / Strand
PAM-1 / AACGGCATCAAGGTGAACTT / NAA / +
PAM-2 / GCCGACAAGCAGAAGAACGG / NAT / +
PAM-3 / ACGGCATCAAGGTGAACTTC / NAG / +
PAM-4 / TCATGGCCGACAAGCAGAAG / NAC / +
PAM-5 / AACTACAACAGCCACAACGT / NTA / +
PAM-6 / AAGAACGGCATCAAGGTGAA / NTT / +
PAM-7 / AGCAGAAGAACGGCATCAAG / NTG / +
PAM-8 / AGAACGGCATCAAGGTGAAC / NTC / +
PAM-9 / GCAGAAGAACGGCATCAAGG / NGA / +
PAM-10 / AAGCAGAAGAACGGCATCAA / NGT / +
PAM-11 / TCAAGGTGAACTTCAAGATC / NGC / +
PAM-12 / AAGGTGAACTTCAAGATCCG / NCA / +
PAM-13 / CAACTACAACAGCCACAACG / NCT / +
PAM-14 / CTTCAAGATCCGCCACAACA / NCG / +
PAM-15 / CATCAAGGTGAACTTCAAGA / NCC / +
PAM-16 / ACAAGTTCAGCGTGTCCGGC / NAG / +
PAM-17 / CCCGCGCCGAGGTGAAGTTC / NAG / +
PAM-18 / TCAAGATCCGCCACAACATC / NAG / +
PAM-19 / TTCAGCGTGTCCGGCGAGGG / NGA / +
PAM-20 / ACCCGCGCCGAGGTGAAGTT / NGA / +
PAM-21 / AAGATCCGCCACAACATCGA / NGA / +

Table S3. Oligonucleotide sequences.

Primer name / Primer sequence(5’-3’)
U6-F / GAGGGCCTATTTCCCATGATTC
GFP-seq F / GCTTGGCACTTGATGTAATTCTC
GFP-seq R / CATGCGGATCCTTCGAACT

Sequencing primers for plasmid construction and GFP mutation.

Oligonucleotide sequences for sgRNA architecture

Primer name / Primer sequence(5’-3’)
NGG-0-F / AAACCAAGTTCAGCGTGTCCGGCGC
NGG-0-R / CACCGCGCCGGACACGCTGAACTTG
NGG-1-F / CACCGCAAGTTCAGCGTGTCCGGCG
NGG-1-R / AAACCGCCGGACACGCTGAACTTGC
NGG-2-F / CACCGCGCGCCGAGGTGAAGTTCGA
NGG-2-R / AAACTCGAACTTCACCTCGGCGCGC
NGG-3-F / CACCGCAAGATCCGCCACAACATCG
NGG-3-F / AAACCGATGTTGTGGCGGATCTTGC
PAM-1-F / CACCGAACGGCATCAAGGTGAACTT
PAM-1-R / AAACAAGTTCACCTTGATGCCGTTC
PAM-2-F / CACCGGCCGACAAGCAGAAGAACGG
PAM-2-R / AAACCCGTTCTTCTGCTTGTCGGCC
PAM-3-F / CACCGACGGCATCAAGGTGAACTTC
PAM-3-R / AAACGAAGTTCACCTTGATGCCGTC
PAM-4-F / CACCGTCATGGCCGACAAGCAGAAG
PAM-4-R / AAACCTTCTGCTTGTCGGCCATGAC
PAM-5-F / CACCGAACTACAACAGCCACAACGT
PAM-5-R / AAACACGTTGTGGCTGTTGTAGTTC
PAM-6-F / CACCGAAGAACGGCATCAAGGTGAA
PAM-6-R / AAACTTCACCTTGATGCCGTTCTTC
PAM-7-F / CACCGAGCAGAAGAACGGCATCAAG
PAM-7-R / AAACCTTGATGCCGTTCTTCTGCTC
PAM-8-F / CACCGAGAACGGCATCAAGGTGAAC
PAM-8-R / AAACGTTCACCTTGATGCCGTTCTC
PAM-9-F / CACCGGCAGAAGAACGGCATCAAGG
PAM-9-R / AAACCCTTGATGCCGTTCTTCTGCC
PAM-10-F / CACCGAAGCAGAAGAACGGCATCAA
PAM-10-R / AAACTTGATGCCGTTCTTCTGCTTC
PAM-11-F / CACCGTCAAGGTGAACTTCAAGATC
PAM-11-R / AAACGATCTTGAAGTTCACCTTGAC
PAM-12-F / CACCGAAGGTGAACTTCAAGATCCG
PAM-12-R / AAACCGGATCTTGAAGTTCACCTTC
PAM-13-F / CACCGCAACTACAACAGCCACAACG
PAM-13-R / AAACCGTTGTGGCTGTTGTAGTTGC
PAM-14-F / CACCGCTTCAAGATCCGCCACAACA
PAM-14-R / AAACTGTTGTGGCGGATCTTGAAGC
PAM-15-F / CACCGCATCAAGGTGAACTTCAAGA
PAM-15-R / AAACTCTTGAAGTTCACCTTGATGC
PAM-16-F / CACCGACAAGTTCAGCGTGTCCGGC
PAM-16-R / AAACGCCGGACACGCTGAACTTGTC
PAM-17-F / CACCGCCCGCGCCGAGGTGAAGTTC
PAM-17-R / AAACGAACTTCACCTCGGCGCGGGC
PAM-18-F / CACCGTCAAGATCCGCCACAACATC
PAM-18-R / AAACGATGTTGTGGCGGATCTTGAC
PAM-19-F / CACCGTTCAGCGTGTCCGGCGAGGG
PAM-19-R / AAACCCCTCGCCGGACACGCTGAAC
PAM-20-F / CACCGACCCGCGCCGAGGTGAAGTT
PAM-20-R / AAACAACTTCACCTCGGCGCGGGTC
PAM-21-F / CACCGAAGATCCGCCACAACATCGA
PAM-21-R / AAACTCGATGTTGTGGCGGATCTTC

qPCR primers used tomeasure the copy number of GFPgene in 293-SC1 cells

Primer name / Primer sequence(5’-3’)
ACTB-F / GTACAGGTCTTTGCGGATGT
ACTB-R / GCTAAGTCCTGCCCTCATTT
FRMD7-F / CAGGTCAGCAGGTTGGTATT
FRMD7-R / CTTGTCCTTTCCTCTGCTCTAAT
GFP-F / TCAAGATCCGCCACAACATC
GFP-R / GTGCTCAGGTAGTGGTTGTC