Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9

March Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9 Luqing Zhang 0 1 2 Ruirui Jia 0 1 2 Norberto J. Palange 0 1 2 Achim Cchitvsanzwhoh Satheka 0 1 2 Jacques Togo 0 1 2 Yao An 0 1 2 Mabwi Humphrey 0 1 2 Luying Ban 0 1 2 Yan Ji 0 1 2 Honghong Jin 0 1 2 Xuechao Feng 0 1 2 Yaowu Zheng 0 1 2 0 1 Transgenic Research Center, School of Life Sciences, Northeast Normal University , Changchun , China , 2 Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University , Changchun , China 1 Data Availability Statement: All relevant data are within the paper 2 Academic Editor: Vincenzo Coppola, Ohio State University Comprehensive Cancer Center , UNITED STATES ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and -galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies. - Funding: This work was supported, in whole or in part, by grants from the National Natural Science Foundation of China (31301189 and 81270953) http:// www.nsfc.gov.cn/Portal0/default152.htm; and the Fundamental Research Funds for the Central Universities (12QNJJ015 and 10JCXK001); and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20130043120010); and the Science-Technology Foundation for Young Scientist of Jilin Province (20130522003JH).The Genetically modified mice represent a powerful tool for dichotomizing gene functions [1,2]. Traditionally, mice carrying targeted mutations are generated by homologous recombination [3]. The technology takes the advantage of cultured embryonic stem cells and chimera generation. The procedures are tedious, less cost-effective and time-consuming. Zinc-finger nucleases (ZFN) [4], transcription activator-like effector nucleases (TALENs) [5,6] and CRISPR/Cas9 system are recently developed technologies in genomic editing. Engineered ZFNs, TALENs and CRISPR/Cas9 have been successfully used to direct site-specific cleavage and mediate Competing Interests: The authors have declared that no competing interests exist. error-prone non-homologous end-joining (NHEJ) or precise homologous recombination (HR) when a DONOR DNA is provided [7,8]. However, ZFN is very tedious in finding efficient DNA binding blocks which requires experiences and TALENs are time-consuming in building the DNA binding domains. Both systems use an artificial nuclease domain (FokI) to make double-stranded breaks (DSBs). CRISPR/Cas9 system is the most updated and the most easily applicable tools. In this system, genome editing is achieved through the bacterial type II clustered regularly interspaced short palindromic repeats (CRISPR) [911] and CRISPR-associated protein 9 (Cas9), a very efficient nuclease itself, to target interested genes and to make specific double-strand breaks (DSBs) [12]. The only required engineering is a 20nt target-complementary CRISPR RNA (crRNA) with the target DNA sequence sitting upstream of a 5protospacer adjacent motif (PAM) site [13]. Various organisms including zebrafish [14], mouse [1], monkey [15], rat [16] and human cells have been successfully modified [17,18]. It has been a major problem for genomic editing that involves large DNA fragment insertion, deletion or replacement, where the larger the fragment, the lower the recombination efficiency [19]. This results in a fact that many reports having modified fragment size around 1kb. However in many cases, large genomic sequence changes are required, for example, deletion of gene clusters, removal of long non-coding RNAs (lncRNA) and swapping of regulatory sequences. Different technologies have been developed to tackle this problem. For examples, BAC and YAC systems have been used to target relatively large DNA fragments. But the efficiency is far from satisfactory. Recently, Xiao and coworkers have reported cleavage of genomic sequence of up to 1Mb in zebrafish by applying TALENs and CRISPR/Cas9 [20]. In this report, two circular plasmids expressing sgRNA and Cas9 were co-injected into mouse zygote to minimize the laborious and extra-careful preparation of in vitro transcribed Cas9 mRNA and sgRNAs [21], leading to complete deletion of the entire Dip2a gene, a 65kb fragment. When the same two circular plasmids were co-transfected with a DONOR plasmid containing selective marker, the 65kb-Dip2a fragment was successfully replaced with a NEO cassette at high frequency in ES cells. A 5.3kb fragment of lacZ reporter gene and NEO cassette was also successfully inserted into downstream of Dip2a promoter by both direct injection and ES cell transfection with high efficiency. The Institutional Animal Care and Use Committee or Animal Experimental Ethics Committee of Northeast Normal University (NENU/IACUC) has specifically approved the entire study, the approval # is AP2013011. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health as well. 129S1/SvJ, C57BL/6J, DBA/2 and CD1 mice were purchased from Vital River (A Charles River Company, Beijing, China). All animals were maintained in a clean facility in Northeast Normal University. Mice were kept in IVC cages (5 per cage) with free access of food and water, under pathogen-free conditions in a room maintained at 20C and 50 20% relative humidity, and under a 12:12-h light:dark cycles. Mice were anesthetized before sacrificing with 1% pelltobarbitalum natricum at a dose of 10 mg/kg. All animal experiments were performed using proper anesthesia before perfusion or any procedures. Plasmid pX330 (http://www.addgene.org/42230/) [18] and pRosa26-1 were obtained from Addgene. pX330 is gifted from Dr. Feng Z (...truncated)