已入深夜,您辛苦了!由于当前在线用户较少,发布求助请尽量完整的填写文献信息,科研通机器人24小时在线,伴您度过漫漫科研夜!祝你早点完成任务,早点休息,好梦!

An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9

清脆的 生物 Cas9 遗传学 空等位基因 终止密码子 阅读框 秀丽隐杆线虫 突变体 基因 基因组编辑 遗传筛选 计算生物学 打开阅读框 肽序列
作者
Han Wang,Heenam Park,Jonathan Liu,Paul W. Sternberg
出处
期刊:G3: Genes, Genomes, Genetics [Oxford University Press]
卷期号:8 (11): 3607-3616 被引量:72
标识
DOI:10.1534/g3.118.200662
摘要

Abstract Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
大幅提高文件上传限制,最高150M (2024-4-1)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
CKK发布了新的文献求助10
1秒前
accept发布了新的文献求助10
1秒前
杉进完成签到 ,获得积分10
1秒前
7秒前
可爱的函函应助adinike采纳,获得10
8秒前
accept完成签到,获得积分10
9秒前
12秒前
嗯哼应助daihq3采纳,获得20
13秒前
汉堡包应助相悦采纳,获得10
13秒前
受伤雁荷发布了新的文献求助10
14秒前
19秒前
AAA111122完成签到,获得积分10
20秒前
21秒前
TJN完成签到 ,获得积分10
22秒前
窦房结完成签到 ,获得积分10
23秒前
秋梨膏完成签到,获得积分10
23秒前
壮观的谷冬完成签到 ,获得积分10
24秒前
24秒前
adinike发布了新的文献求助10
24秒前
ylf发布了新的文献求助10
26秒前
飞儿随缘发布了新的文献求助10
28秒前
koitoyu完成签到,获得积分10
29秒前
你好好好完成签到,获得积分10
30秒前
乐乐完成签到 ,获得积分10
30秒前
31秒前
笨笨西牛完成签到 ,获得积分10
34秒前
Aiden发布了新的文献求助10
35秒前
欣慰煎饼发布了新的文献求助10
35秒前
微笑的砖头完成签到,获得积分10
36秒前
37秒前
38秒前
39秒前
飞儿随缘完成签到,获得积分10
39秒前
39秒前
xiaomeng完成签到 ,获得积分10
40秒前
Robin发布了新的文献求助10
42秒前
炸炸桃发布了新的文献求助10
43秒前
千纸鹤发布了新的文献求助10
43秒前
磨磨唧唧应助沉淀采纳,获得30
47秒前
852应助沉淀采纳,获得10
47秒前
高分求助中
歯科矯正学 第7版(或第5版) 1004
Semiconductor Process Reliability in Practice 1000
Smart but Scattered: The Revolutionary Executive Skills Approach to Helping Kids Reach Their Potential (第二版) 1000
Nickel superalloy market size, share, growth, trends, and forecast 2023-2030 600
GROUP-THEORY AND POLARIZATION ALGEBRA 500
Mesopotamian divination texts : conversing with the gods : sources from the first millennium BCE 500
Days of Transition. The Parsi Death Rituals(2011) 500
热门求助领域 (近24小时)
化学 医学 生物 材料科学 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 基因 遗传学 催化作用 物理化学 免疫学 量子力学 细胞生物学
热门帖子
关注 科研通微信公众号,转发送积分 3234378
求助须知:如何正确求助?哪些是违规求助? 2880736
关于积分的说明 8216789
捐赠科研通 2548319
什么是DOI,文献DOI怎么找? 1377665
科研通“疑难数据库(出版商)”最低求助积分说明 647925
邀请新用户注册赠送积分活动 623304