CRISPR-based technologies have emerged as powerful tools to alter genomes and mark chromosomal loci, but an inexpensive method for generating large numbers of RNA guides for whole genome screening and labeling is lacking. Using a method that permits library construction from any source of DNA, we generated guide libraries that label repetitive loci or a single chromosomal locus in Xenopus egg extracts and show that a complex library can target the E. coli genome at high frequency.
Bibliographical noteFunding Information:
We thank the Doudna lab (UC Berkeley) for providing us with a dCas9 cDNA, as well as advice on bacterial expression and purification, and Adam Session and Dan Rokhsar for sequence information regarding X. laevis repeats. This work was supported by NIH R01GM098766 (R.H.) and NIH S10RR26758 (T.W.). A.W.G. was supported by the National Science Foundation Graduate Research Fellowship Program. A patent is in preparation on CRISPR-EATING (R.H. and A.B.L.). mNeonGreen DNA sequence is licensed under MTA from Allele Biotechnology and Pharmaceuticals to the Regents of the University of California.
© 2015 Elsevier Inc.