Ha Lab reveals mechanisms of increased Cas9s specificity using single-molecule FRET

10/9/2018 9:40:54 AM CPLC Staff

Image from: Nature Structural & Molecular Biology  ISSN 1545-9985 (online)

RNA-programmable CRISPR enzymes are used for wide-ranging applications including genome engineering. CRISPR nucleases in complex with a guide-RNA searches for its target DNA, unwinds it, and if the DNA sequence matches the RNA sequence over a critical length, cleaves the DNA at a specific location. How the CRISPR system can identify a correct target rapidly and accurately is an important question that has implications on potentially increasing the on-target cleavage activity and decreasing the off-target cleavage activity. CPLC student Digvijay Singh in the group of Prof. Taekjip Ha, in collaboration with the Doudna lab at UC Berkeley, developed single-molecule fluorescence measurement technologies to perform comparative analysisof CRISPR-enzymes, the most widely used Cas9, its engineered derivatives (EngCas9), and Cpf1. The group found that all Cas9s, including EngCas9s, require only 9-10 base pair (bp) matches for stable binding and ~16 bp match for cleavage. Cpf1 was significantly more specific in binding, requiring ~17 bp. Singh also developed a single molecule FRET DNA unwinding assay and showed that Cas9 cleaves DNA only from the maximally unwound DNA state. Partial sequence mismatches that perturb cleavage also showed reduction of DNA unwinding, confirming that DNA unwinding is a critical checkpoint for cleavage of the correct target. EngCas9s mutations destabilize the unwound state thereby reducing its extent and lifetime, even for DNA with very few sequence mismatches, contributing to the enhanced specificity of cleavage. Following proofreading, EngCas9s nuclease domains also take longer to activate and cleave. All of these form the basis of improved cleavage specificity of EngCas9s.

1/24/2014 The Climate Corporation

Software Engineer/CS Intern

The Climate Corporation is looking for talented and motivated Computer Science interns for several of our
engineering teams. As a CS intern, you will be responsible for designing, building, and testing new products
and services in a fast-paced, iterative development environment.
We leverage big data and cloud computing to build truly innovative products that are transforming industries
and protecting livelihoods every day. This mission translates into hard and fun engineering challenges —
solving complex computational problems, managing massive data sets and applying techniques from multiple

What you offer:

  • Initiative to figure out how things work and to push the limits of your own knowledge
  • Write well-structured, easily maintainable, well-documented code
  • Able to collaborate with other engineers on design and development of challenging projects
  • Great debugging and reasoning skills
  • Demonstrated passion for quality


  • Intellectual drive to learn anything
  • Pursuing or completed a Bachelors in computer science, computer engineering, or software
  • engineering
  • Strong working knowledge at least one programming language
  • Stellar academic record or equivalent work experience
  • Knowledge of software engineering or server-side Internet technologies
  • Familiarity with command-line tools on a Unix shell
  • A desire to work on hard problems
  • At this time we are only offering internships to candidates that have the legal right to work in the US.

What we offer:

  • Valuable internship experience at a well-known and fast-growing start-up
  • Opportunity to work with large datasets, help build features in and tools around high-throughput, lowlatency
  • Pricing and Settlement systems
  • Opportunity to learn bleeding-edge technologies, such as Hadoop, NoSQL databases, functional
  • programming languages like Clojure, and contribute your work back to open-source projects
  • Mentoring by smart, passionate software engineers
  • Energetic, idea-driven and transparent work environment
  • We provide meals and a large assortment of snacks, drinks, fruits, coffees, and teas to help you get
  • through the day
  • We sponsor hackathons, meet up groups, networking events, tech-talks, and conference trips. If you
  • want to get involved in the tech community, we'll support your efforts
  • The internship is paid, with flexible Winter, Spring or Summer start dates. You must be available for
  • full-time commitment for the course of the internship (40 hours/week).
  • www.climate.com/careers

To Apply: https://hire.jobvite.com/j?bj=okOTXfwI&s=U_Illinois-_U/C

Teams Hiring Computer Science Interns
Apps and Preso - (Agent Applications TWI/API)
Help agents and growers save time and make more money by delivering an integrated, world class
technology suite to help them better manage and grow their business.

Climate.com (Grower Application)
Provide growers with tools and insights that help them make smarter, more informed decisions about
their operations.

Protect Back End – Insurance Policy Management
Build world-class products and tools to help growers and agents mitigate risk against weather and other

Risk & Economics – Policy Modeling and Portfolios
Produce and evaluate aggregated portfolio metrics by providing and supporting a comprehensive,
robust, and automated system.

Blue Marble – Data Ingestion and Transformation
Deliver accurate agriculture, weather, geoscience, and climate related data and metadata.

Platform – Storage and Computer Services
Provide scalable tools and services to support large-scale agricultural and risk management products.

Structures – Yield Modeling
Predict the economic impact of weather

To Apply: https://hire.jobvite.com/j?bj=okOTXfwI&s=U_Illinois-_U/C