Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion

Abstract

Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

“…Cas12a has several advantages over Cas9 for in vitro DNA editing, thus it could serve as a great supplement to current methods. For instance, Cas12a cleaves double‐stranded DNA to produce relatively consistent sticky ends rather than uncertainly 1–3 bp sticky ends (Shou, Li, Liu, & Wu, ), a trans‐acting crRNA is not required thus the crRNA is shorter (~42 bp), and it can process its own crRNA array into mature crRNA to enable easy multiplex gene editing (Fonfara, Richter, Bratovič, Le Rhun, & Charpentier, ; P. Gao, Yang, Rajashankar, Huang, & Patel, ). These characteristics facilitate the utilization of Cas12a as a DNA editing tool in vitro.…”

Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection

“…We performed CBS insertions by DNA-fragment editing and screened for single-cell CRISPR clones (Fig. 1c, d) [37,38]. We first inserted single ("F") or tandem ("FF") forward-oriented CBS elements into the location between the Pcdhα cluster and its HS5-1 enhancer ( Fig.…”

“…The preparation of sgRNA pairs and Cas9 mRNA was recently described [38]. Briefly, to obtain sgRNAs for microinjection of zygotes, we performed in vitro transcription using DNA templates generated by PCR with a forward primer containing a T7 promoter followed by targeting sequences and a common reverse primer.…”

“…The plasmids of sgRNAs for cell transfection experiments were constructed as previously described [37,38]. Briefly, pairs of complementary oligonucleotides for generating sgRNAs (Additional file 2: Table S1) were annealed with 5′ overhangs of "ACCG" and "AAAC," and cloned into a BsaI-linearized pGL3 vector under the control of the U6 promoter.…”

“…Here, by a combination of CRISPR DNA-fragment editing [37,38] and chromosome conformation capture [3] experiments as well as Bayesian modeling, we show that ectopic and endogenous CTCF sites function as topological insulators in an orientation-independent manner through CTCF-mediated directional chromatin looping throughout the mammalian genome. In addition, genetic experiments, in conjunction with computational polymer simulation of cohesin loop extrusion, demonstrate that tandem-arrayed CTCF sites ensure stochastic spatial accessibility of repertoires of promoters and their balanced usage.…”