Transgene-free genome editing by germline injection of CRISPR/Cas RNA.
Genome modification by CRISPR/Cas offers its users the ability to target endogenous sites in the genome for cleavage and for engineering precise genomic changes using template-directed repair, all with unprecedented ease and flexibility of targeting. As such, CRISPR/Cas is just part of a set of recently developed and rapidly improving tools that offer great potential for researchers to functionally access the genomes of organisms that have not previously been extensively used in a laboratory setting. We describe in detail protocols for using CRISPR/Cas to target genes of experimental organisms, in a manner that does not require transformation to obtain transgenic lines and that should be readily applicable to a wide range of previously little-studied species.
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA; Howard Hughes Medical Institute, Pasadena, California, USA.
Hillel T
HT
Schwartz
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA; Howard Hughes Medical Institute, Pasadena, California, USA. Electronic address: pws@caltech.edu.
Paul W
PW
Sternberg
D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D017173:Caenorhabditis elegans / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D016678:Genome; D005854:Germ Cells / Q000378:metabolism; D016296:Mutagenesis; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000008:administration & dosage / Q000235:genetics; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics; D014158:Transcription, Genetic; D019076:Transgenes
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger; D004720:Endonucleases
CRISPR; Caenorhabditis elegans; Cas9; Engineered nuclease; Gene targeting; Germline injection; Nematode; RNA injection; Transgene-free